# Patent

## Title
Method for simultaneous production of multiple proteins; vectors and cells for use therein

## Abstract

Described is the production of proteins in a host cell. Even more specifically, described are methods for improving expression of two or more proteins in a cells or host cell. The methods are suited for production of, for example, recombinant antibodies that can be used in pharmaceutical preparations or as diagnostic tools. In certain embodiments, provided are methods for obtaining a cell that expresses two or more proteins comprising providing the cell with two or more protein expression units encoding two or more proteins, characterized in that at least two of the protein expression units comprise at least one STAR sequence.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional application of U.S. patent application Ser. No. 11/013,031, filed Dec. 14, 2004, now U.S. Pat. No. 7,364,878, which is a continuation application of International Patent Application No. PCT/NL03/00432, filed Jun. 13, 2003, designating the United States, and published in English as International Patent Publication No. WO 03/106684 on Dec. 24, 2003, which claims benefit, under the Paris Convention, of European Patent Application No. EP 02077350 filed Jun. 14, 2002, the entirety of each of which are hereby incorporated by reference.
STATEMENT ACCORDING TO 37 C.F.R. §1.52(e)(5)—SEQUENCE LISTING SUBMITTED ON COMPACT DISC
Pursuant to 37 C.F.R. §1.52(e)(1)(iii), a compact disc containing an electronic version of the Sequence Listing has been submitted concomitant with this application, the contents of which are hereby incorporated by reference. A second compact disk is submitted and is an identical copy of the first compact disc. The discs are labeled, “copy 1” and “copy 2,” respectively, and each disc contains one file entitled “P60556PC00.txt” which is 502 KB, and created on Nov. 29, 2004.


TECHNICAL FIELD
The invention relates to the fields of biochemistry, molecular biology, pharmacology and diagnosis. More specifically, the invention relates to the production of proteins in a host cell. Even more specifically, the invention relates to a method for improving expression of two or more proteins in a cells or host cell. The method is suited for production of, for example, recombinant antibodies that can be used in a pharmaceutical preparation or as a diagnostic tool.
BACKGROUND OF THE INVENTION
Proteins are produced in systems for a wide range of applications in biology and biotechnology. These include research into cellular and molecular function, production of proteins as biopharmaceuticals or diagnostic reagents, and modification of the traits or phenotypes of livestock and crops. Biopharmaceuticals are usually proteins that have an extracellular function, such as antibodies for immunotherapy or hormones or cytokines for eliciting a cellular response. Proteins with extracellular functions exit the cell via the secretory pathway, and undergo post-translational modifications during secretion. The modifications (primarily glycosylation and disulfide bond formation) do not occur in bacteria. Moreover, the specific oligosaccharides attached to proteins by glycosylating enzymes are species and cell-type specific. These considerations often limit the choice of host cells for heterologous protein production to eukaryotic cells (Kaufman, 2000). For expression of human therapeutic proteins, host cells such as bacteria, yeast, or plants may be inappropriate. Even the subtle differences in protein glycosylation between rodents and human, for example, can be sufficient to render proteins produced in rodent cells unacceptable for therapeutic use (Sheeley et al., 1997). The consequences of improper (i.e., non-human) glycosylation include immunogenicity, reduced functional half-life, and loss of activity. This limits the choice of host cells further, to human cell lines or to cell lines such as Chinese Hamster Ovary (CHO) cells, which may produce glycoproteins with human-like carbohydrate structures (Liu, 1992).
Some proteins of biotechnological interest are functional as multimers, i.e., they consist of two or more, possibly different, polypeptide chains in their biologically and/or biotechnologically active form. Examples include antibodies (Wright & Morrison, 1997), bone morphogenetic proteins (Groeneveld & Burger, 2000), nuclear hormone receptors (Aranda & Pascual, 2001), heterodimeric cell surface receptors (e.g., T cell receptors, (Chan & Mak, 1989)), integrins (Hynes, 1999), and the glycoprotein hormone family (chorionic gonadotrophin, pituitary luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone, (Thotakura & Blithe, 1995)). Production of such multimeric proteins in heterologous systems is technically difficult due to a number of limitations of current expression systems. These limitations include (1) difficulties in isolating recombinant cells/cell lines that produce the monomer polypeptides at high levels (predictability and yield), (2) difficulties in attaining production of the monomeric polypeptides in stoichiometrically balanced proportions (Kaufman, 2000), and (3) declines in the levels of expression during the industrial production cycle of the proteins (stability). These problems are described in more detail below.
(1) Recombinant proteins such as antibodies that are used as therapeutic compounds need to be produced in large quantities. The host cells used for recombinant protein production must be compatible with the scale of the industrial processes that are employed. Specifically, the transgene (or the gene encoding a protein of interest, the two terms are used interchangeably herein) expression system used for the heterologous protein needs to be retained by the host cells in a stable and active form during the growth phases of scale-up and production. This is achieved by integration of the transgene into the genome of the host cell. However, creation of recombinant cell lines by conventional means is a costly and inefficient process due to the unpredictability of transgene expression among the recombinant host cells. The unpredictability stems from the high likelihood that the transgene will become inactive due to gene silencing (McBurney et al., 2002). Using conventional technologies, the proportion of recombinant host cells that produce one polypeptide at high levels ranges from 1-2%. In order to construct a cell line that produces two polypeptides at high levels, the two transgenes are generally integrated independently. If the two transgenes are transfected simultaneously on two separate plasmids, the proportion of cells that will produce both polypeptides at high levels will be the arithmetic product of the proportions for single transgenes. Therefore, the proportion of such recombinant cell lines ranges from one in 2,500 to one in 10,000. For multimeric proteins with three or more subunits, the proportions decline further. These high-producing cell lines must subsequently be identified and isolated from the rest of the population. The methods required to screen for these rare high-expressing cell lines are time-consuming and expensive.
An alternative to simultaneous transfection of two transgene-bearing plasmids is sequential transfection. In this case the proportion of high-yielding clones will be the sum of the proportions for single transgenes, i.e., 2-4%. Sequential transfection however has (major) drawbacks, including high costs and poor stability. The high costs result from various factors: in particular, the time and resources required for screening for high-expressing cell lines is doubled, since high expression of each subunit must be screened for separately. The poor overall stability of host cells expressing two polypeptides is a consequence of the inherent instability of each of the two transgenes.
(2) Production of multimeric proteins requires balanced levels of transcriptional and translational expression of each of the polypeptide monomers. Imbalanced expression of the monomers is wasteful of the costly resources used in cell cultivation. Moreover, the imbalanced expression of one monomer can have deleterious effects on the cell. These effects include (a) sequestration of cellular factors required for secretion of the recombinant proteins (e.g., chaperones in the endoplasmic reticulum, (Chevet et al., 2001)), and (b) induction of stress responses that result in reduced rates of growth and protein translation, or even in apoptosis (programmed cell death) (Pahl & Baeuerle, 1997, Patil & Walter, 2001). These deleterious effects lead to losses in productivity and yield and to higher overhead costs.
(3) Silencing of transgene expression during prolonged host cell cultivation is a commonly observed phenomenon. In vertebrate cells it can be caused by formation of heterochromatin at the transgene locus, which prevents transcription of the transgene. Transgene silencing is stochastic; it can occur shortly after integration of the transgene into the genome, or only after a number of cell divisions. This results in heterogeneous cell populations after prolonged cultivation, in which some cells continue to express high levels of recombinant protein while others express low or undetectable levels of the protein (Martin & Whitelaw, 1996, McBurney et al., 2002). A cell line that is used for heterologous protein production is derived from a single cell, yet is often scaled up to, and maintained for long periods at, cell densities in excess of ten million cells per milliliter in cultivators of 1,000 liters or more. These large cell populations (1014-1016 cells) are prone to serious declines in productivity due to transgene silencing (Migliaccio et al., 2000, Strutzenberger et al., 1999).
The instability of expression of recombinant host cells is particularly severe when transgene copy numbers are amplified in an attempt to increase yields. Transgene amplification is achieved by including a selectable marker gene such as dihydrofolate reductase (DHFR) with the transgene during integration. Increased concentrations of the selection agent (in the case of DHFR, the drug methotrexate) select for cells that have amplified the number of DHFR genes in the chromosome. Since the transgene and DHFR are co-localized in the chromosome, the transgene copy number increases too. This is correlated with an increase in the yield of the heterologous protein (Kaufman, 1990). However, the tandem repeats of transgenes that result from amplification are highly susceptible to silencing (Garrick et al., 1998, Kaufman, 1990, McBurney et al., 2002). Silencing is often due to a decline in transgene copy number after the selection agent is removed (Kaufman, 1990). Removal of the selection agent, however, is routine during industrial biopharmaceutical production, for two reasons. First, cultivation of cells at industrial scales in the presence of selection agents is not economically feasible, as the agents are expensive compounds. Second, and more importantly, concerns for product purity and safety preclude maintaining selection during a production cycle. Purifying a recombinant protein and removing all traces of the selection agent is necessary if the protein is intended for pharmaceutical use. However, it is technically difficult and prohibitively expensive to do so, and demonstrating that this has been achieved is also difficult and expensive. Therefore, amplification-based transgenic systems that require continual presence of selection agents are disadvantageous.
Alternatively, silencing can be due to epigenetic effects on the transgene tandem repeats, a phenomenon known as Repeat Induced Gene Silencing (RIGS) (Whitelaw et al., 2001). In these cases the copy number of the transgene is stable, and silencing occurs due to changes in the chromatin structure of the transgenes (McBurney et al., 2002). The presence of a selection agent during cell cultivation may be unable to prevent silencing of the transgene transcription unit because transgene expression is independent of expression of the selectable marker. The lack of a means to prevent RIGS in conventional transgenic systems thus results in costly losses in productivity.
SUMMARY OF THE INVENTION
The problems associated with conventional transgene expression technologies for protein production and more specifically for multimeric protein production demonstrate a need in the art for a system that overcomes these problems. Described is a system for creating cells or host cells/cell lines that efficiently express two or more proteins, for example, two or more polypeptide monomers and optionally produce functional multimeric proteins from them. Important examples of heterologous multimer proteins are recombinant antibodies. In certain embodiments, the invention takes advantage of proprietary DNA elements that protect transgenes from silencing, termed STabilizing Anti-Repressor (STAR or STARTM; these terms will be used interchangeably herein) elements, for the production of two or more proteins.
The invention also discloses a novel configuration of transcriptional and translational elements and selectable marker genes. In certain embodiments, the invention uses antibiotic resistance genes and protein translation initiation sites with reduced translation efficiency (for example, an Internal Ribosome Entry Site, IRES) in novel ways that improve heterologous protein expression. The combination of the STAR elements and these other elements results in a system for obtaining a cell which expresses two or more proteins that (1) predictably produces a high proportion of recombinant cell lines with high yields of heterologous proteins, (2) exhibits balanced and proportional expression of two or more polypeptide monomers which are constituents of a multimeric protein, and (3) creates recombinant cell lines with stable productivity characteristics.
Therefore, provided is in certain embodiments, a method for obtaining a cell which expresses two or more proteins comprising providing the cell with two or more protein expression units encoding the two or more proteins, characterized in that at least two of the protein expression units comprise at least one STAR sequence.
STAR-sequences can be identified (as disclosed, for example, in Example I of EP 01202581.3) using a method of detecting, and optionally selecting, a DNA sequence with a gene transcription-modulating quality, comprising providing a transcription system with a variety of fragment-comprising vectors, the vectors comprising i) an element with a gene-transcription repressing quality, and ii) a promoter directing transcription of a reporter gene, the method further comprising performing a selection step in the transcription system in order to identify the DNA sequence with the gene transcription modulating quality. In certain embodiments, the fragments are located between i) the element with a gene-transcription repressing quality, and ii) the promoter directing transcription of the reporter gene. RNA polymerase initiates the transcription process after binding to a specific sequence, called the promoter, which signals where RNA synthesis should begin. A modulating quality can enhance transcription from the promoter in cis, in a given cell type and/or a given promoter. The same DNA sequence can comprise an enhancing quality in one type of cell or with one type of promoter, whereas it can comprise another or no gene transcription modulating quality in another cell or with another type of promoter. Transcription can be influenced through a direct effect of the regulatory element (or the protein(s) binding to it) on the transcription of a particular promoter. Transcription can however, also be influenced by an indirect effect, for instance because the regulatory element affects the function of one or more other regulatory elements. A gene transcription modulating quality can also comprise a stable gene transcription quality. With stable is meant that the observed transcription level is not significantly changed over at least 30 cell divisions. A stable quality is useful in situations wherein expression characteristics should be predictable over many cell divisions. Typical examples are cell lines transfected with foreign genes. Other examples are transgenic animals and plants and gene therapies. Very often, introduced expression cassettes function differently after increasing numbers of cell divisions or plant or animal generations. In certain embodiments, a stable quality comprises a capacity to maintain gene transcription in subsequent generations of a transgenic plant or animal. Of course, if expression is inducible, this quality comprises the quality to maintain inducibility of expression in subsequent generations of a transgenic plant or animal. Frequently, expression levels drop dramatically with increasing numbers of cell divisions. With the herein described method for identification of a DNA sequence with a gene transcription modulating quality, it is possible to detect and optionally select a DNA sequence that is capable of at least in part preventing the dramatic drop in transcription levels with increasing numbers of cell divisions. In certain embodiments, the gene transcription modulating quality comprises a stable gene transcription quality. Strikingly, fragments comprising a DNA sequence with the stable gene transcription quality can be detected and optionally selected with the method for identification of a DNA sequence with a gene transcription modulating quality, in spite of the fact that this method does not necessarily measure long term stability of transcription. In certain embodiments, this gene transcription modulating quality comprises a stable gene transcription enhancing quality. It has been observed that incorporation of a DNA sequence with a gene transcription modulating quality in an expression vector with a gene of interest, results in a higher level of transcription of the gene of interest, upon integration of the expression vector in the genome of a cell and moreover that the higher gene expression level is also more stable than in the absence of the DNA sequence with a gene transcription modulating quality.
In experiments designed to introduce a gene of interest into the genome of a cell and to obtain expression of the gene of interest, the following has been observed. If together with the gene of interest also a DNA sequence with a gene transcription modulating quality was introduced, more clones could be detected that expressed more than a certain amount of gene product of the gene of interest, than when the DNA sequence was not introduced together with the gene of interest. Thus an identified DNA sequence with gene transcription modulating quality also provides a method for increasing the number of cells expressing more than a certain level of a gene product of a gene of interest upon providing the gene of interest to the genome of the cells, comprising providing the cell with a DNA sequence comprising a gene transcription modulating quality together with the gene of interest.
The chances of detecting a fragment with a gene transcription-modulating quality vary with the source from which the fragments are derived. Typically, there is no prior knowledge of the presence or absence of fragments with that quality. In those situations many fragments will not comprise a DNA sequence with a gene transcription-modulating quality. In these situations a formal selection step for DNA sequences with that quality is introduced. This is done by selection vectors comprising the sequence on the basis of a feature of a product of the reporter gene, that can be selected for or against. For instance, the gene product may induce fluorescence or a color deposit (e.g., green fluorescent protein and derivatives, luciferase, or alkaline phosphatase) or confer antibiotic resistance or induce apoptosis and cell death.
A method for the identification of a DNA sequence with a gene transcription modulating quality is particularly suited for detecting and optionally selecting a DNA sequence comprising a gene transcription-enhancing quality. It has been observed that at least some of the selected DNA sequences, when incorporated into an expression vector comprising a gene of interest, can dramatically increase gene transcription of the gene of interest in a host cell even when the vector does not comprise an element with a gene-transcription repressing quality. This gene transcription enhancing quality is very useful in cell lines transfected with foreign genes or in transgenic animals and plants.
The transcription system can be a cell free in vitro transcription system. With the current expertise in automation such cell free systems can be accurate and quick. However, the transcription system preferably comprises host cells. Using host cells warrants that fragments are detected and optionally selected with activity in cells.
An element with a gene transcription repressing quality will repress transcription from a promoter in the transcription system used. Repression does not have to lead to undetectable expression levels. Important is that the difference in expression levels in the absence or presence of repression is detectable and optionally selectable. In certain embodiments, gene-transcription repression in the vectors results in gene-transcription repressing chromatin. In certain embodiments, DNA sequences can be detected, and optionally selected that are capable of at least in part counteracting the formation of gene-transcription repressing chromatin. In one aspect a DNA sequence capable of at least in part counteracting the formation of gene-transcription repressing chromatin comprises a stable gene transcription quality. In certain embodiments, the DNA sequence involved in gene-transcription repression is a DNA sequence that is recognized by a protein complex and wherein the transcription system comprises the complex. In certain embodiments, the complex comprises a heterochromatin-binding protein comprising HP1, a Polycomb-group (Pc-G) protein, a histone deacetylase activity or MeCP2 (methyl-CpG-binding protein). Many organisms comprise one or more of these proteins. These proteins frequently exhibit activity in other species as well. The complex can thus also comprise proteins from two or more species. The mentioned set of known chromatin-associated protein complexes are able to convey long-range repression over many base pairs. The complexes are also involved in stably transferring the repressed status of genes to daughter cells upon cell division. Sequences selected in this way are able to convey long-range anti-repression over many base pairs (van der Vlag et al., 2000).
The vector used can be any vector that is suitable for cloning DNA and that can be used in a transcription system. When host cells are used it is preferred that the vector is an episomally replicating vector. In this way, effects due to different sites of integration of the vector are avoided. DNA elements flanking the vector at the site of integration can have effects on the level of transcription of the promoter and thereby mimic effects of fragments comprising DNA sequences with a gene transcription modulating quality. In certain embodiments, the vector comprises a replication origin from the Epstein-Barr virus (EBV), OriP, and a nuclear antigen (EBNA-1). Such vectors are capable of replicating in many types of eukaryotic cells and assemble into chromatin under appropriate conditions.
DNA sequences with gene transcription modulating quality can be obtained from different sources, for example, from a plant or vertebrate, or derivatives thereof, or a synthetic DNA sequence or one constructed by means of genetic engineering. In certain embodiments, the DNA sequence comprises a sequence as depicted in Table 3 and/or SEQ ID NOs:1-119 and/or a functional equivalent and/or a functional fragment thereof. SEQ ID NOs:1-119 comprise STAR1-STAR65 (SEQ ID NOS:1-65); sequences comprising STAR66 and testing set (SEQ ID NOS:66-84); and sequences comprising Arabidopsis STAR A1-A35 (SEQ ID NOS:85-119) (herein after SEQ ID NOs:1-119).
Several methods are available in the art to extract sequence identifiers from a family of DNA sequences sharing a certain common feature. Such sequence identifiers can subsequently be used to identify sequences that share one or more identifiers. Sequences sharing such one or more identifiers are likely to be members of the same family of sequences, i.e., they are likely to share the common feature of the family. Herein, a large number of sequences comprising STAR activity (so-called STAR sequences or STAR elements) were used to obtain sequence identifiers (patterns) which are characteristic for sequences comprising STAR activity. These patterns can be used to determine whether a test sequence is likely to contain STAR activity. A method for detecting the presence of a STAR sequence within a nucleic acid sequence of about 50-5000 base pairs is thus herein provided, comprising determining the frequency of occurrence in the sequence of at least one sequence pattern and determining that the frequency of occurrence is representative of the frequency of occurrence of at least one sequence pattern in at least one sequence comprising a STAR sequence. In principle any method is suited for determining whether a sequence pattern is representative of a STAR sequence. Many different methods are available in the art. In certain embodiments, the step of determining that the occurrence is representative of the frequency of occurrence of at least one sequence pattern in at least one sequence comprising a STAR sequence comprises, determining that the frequency of occurrence of at least one sequence pattern significantly differs between at least one STAR sequence and at least one control sequence. In principle any significant difference is discriminative for the presence of a STAR sequence. However, in a particularly preferred embodiment, the frequency of occurrence of at least one sequence pattern is significantly higher in at least one sequence comprising a STAR sequence compared to at least one control sequence.
As described above, a considerable number of sequences comprising a STAR sequence have been identified herein. It is possible to use these sequences to test how efficient a pattern is in discriminating between a control sequence and a sequence comprising a STAR sequence. Using so-called discriminant analysis it is possible to determine on the basis of any set of STAR sequences in a species, the most optimal discriminative sequence patters or combination thereof. Thus, preferably, at least one of the patterns is selected on the basis of optimal discrimination between at least one sequence comprising a STAR sequence and a control sequence.
Preferably, the frequency of occurrence of a sequence pattern in a test nucleic acid is compared with the frequency of occurrence in a sequence known to contain a STAR sequence. In this case a pattern is considered representative for a sequence comprising a STAR sequence if the frequencies of occurrence are similar. Even more preferably, another criterion is used. The frequency of occurrence of a pattern in a sequence comprising a STAR sequence is compared to the frequency of occurrence of the pattern in a control sequence. By comparing the two frequencies it is possible to determine for each pattern thus analyzed, whether the frequency in the sequence comprising the STAR sequence is significantly different from the frequency in the control sequence. Then a sequence pattern is considered to be representative of a sequence comprising a STAR sequence, if the frequency of occurrence of the pattern in at least one sequence comprising a STAR sequence is significantly different from the frequency of occurrence of the same pattern in a control sequence. By using larger numbers of sequences comprising a STAR sequence the number of patterns for which a statistical difference can be established increases, thus enlarging the number of patterns for which the frequency of occurrence is representative for a sequence comprising a STAR sequence. In certain embodiments, the frequency of occurrence is representative of the frequency of occurrence of at least one sequence pattern in at least two sequences comprising a STAR sequence, more preferably, in at least five sequences comprising a STAR sequence. More preferably, in at least ten sequences comprising a STAR sequence. More preferably, the frequency of occurrence is representative of the frequency of occurrence of at least one sequence pattern in at least 20 sequences comprising a STAR sequence. Particularly preferred, the frequency of occurrence is representative of the frequency of occurrence of at least one sequence pattern in at least 50 sequences comprising a STAR.
The patterns that are indicative for a sequence comprising a STAR sequence are also dependent on the type of control nucleic acid used. The type of control sequence used is preferably selected on the basis of the sequence in which the presence of a STAR sequence is to be detected. In certain embodiments, the control sequence comprises a random sequence comprising a similar AT/CG content as at least one sequence comprising a STAR sequence. Even more preferably, the control sequence is derived from the same species as the sequence comprising the STAR sequence. For instance, if a test sequence is scrutinized for the presence of a STAR sequence, active in a plant cell, then preferably, the control sequence is also derived from a plant cell. Similarly, for testing for STAR activity in a human cell, the control nucleic acid is preferably also derived from a human genome. In certain embodiments, the control sequence comprises between 50% and 150% of the bases of at least one sequence comprising a STAR sequence. Particularly preferred, the control sequence comprises between 90% and 110% of the bases of at least one sequence comprising a STAR sequence. More preferably, between 95% and 105%.
A pattern can comprise any number of bases larger than two. In certain embodiments, at least one sequence pattern comprises at least five, more preferably, at least six bases. Even more preferably, at least one sequence pattern comprises at least eight bases. In certain embodiments, at least one sequence pattern comprises a pattern listed in Table 4 and/or Table 5. A pattern may consist of a consecutive list of bases. However, the pattern may also comprise bases that are interrupted one or more times by a number of bases that are not, or only, partly discriminative. A partly discriminative base is for instance indicated as a purine.
The presence of STAR activity may be verified using a functional assay. Several methods are presented herein to determine whether a sequence comprises STAR activity. STAR activity is confirmed if the sequence is capable of performing at least one of the following functions: (i) at least in part inhibiting the effect of a sequence comprising a gene transcription repressing element of the invention, (ii) at least in part blocking chromatin-associated repression, (iii) at least in part blocking activity of an enhancer, (iv) conferring upon an operably linked nucleic acid encoding a transcription unit compared to the same nucleic acid alone. (iv-a) a higher predictability of transcription, (iv-b) a higher transcription, and/or (iv-c) a higher stability of transcription over time.
The large number of sequences comprising STAR activity identified herein open up a wide variety of possibilities to generate and identify sequences comprising the same activity in kind, but not necessarily in amount. For instance, it is well within the reach of a skilled person to alter the sequences identified herein and test the altered sequence for STAR activity. Such altered sequences are, therefore, also included herein and can be used in a method for obtaining a cell which expresses two or more proteins or in a method for identifying a cell wherein expression of two or more proteins is in a predetermined ratio. Alteration can include deletion, insertion and mutation of one or more bases in the sequences.
Sequences comprising STAR activity were identified in stretches of 400 bases. However, it is expected that not all of these 400 bases are required to retain STAR activity. Methods to delimit the sequences that confer a certain property to a fragment of between 400 and 5000 bases are well known. The minimal sequence length of a fragment comprising STAR activity is estimated to be about 50 bases.
Tables 4 and 5 list patterns of 6 bases that have been found to be over represented in nucleic acid molecules comprising STAR activity. This over representation is considered to be representative for a STAR sequence. The tables were generated for a family of 65 STAR sequences. Similar tables can be generated starting from a different set of STAR sequences, or from a smaller or larger set of STAR sequences. A pattern is representative for a STAR sequence if it is over represented in the STAR sequence compared to a sequence not comprising a STAR element. This can be a random sequence. However, to exclude a non-relevant bias, the sequence comprising a STAR sequence is preferably compared to a genome or a significant part thereof, more preferably, a genome of a vertebrate or plant, and even more preferably, a human genome. A significant part of a genome is for instance a chromosome. In certain embodiments, the sequence comprising a STAR sequence and the control sequence are derived from a nucleic acid of the same species.
The more STAR sequences are used for the determination of the frequency of occurrence of sequence patterns, the more representative for STARS the patterns are that are over-or under-represented. Considering that many of the functional features that can be displayed by nucleic acids are mediated by proteinaceous molecules binding to it, it is preferred that the representative pattern is over-represented in the STAR sequences. Such an over-represented pattern can be part of a binding site for such a proteinaceous molecule. In certain embodiments, the frequency of occurrence is representative of the frequency of occurrence of at least one sequence pattern in at least two sequences comprising a STAR sequence, more preferably, in at least five sequences comprising a STAR sequence, and even more preferably, in at least ten sequences comprising a STAR sequence. More preferably, the frequency of occurrence is representative of the frequency of occurrence of at least one sequence pattern in at least 20 sequences comprising a STAR sequence. Particularly preferred, the frequency of occurrence is representative of the frequency of occurrence of at least one sequence pattern in at least 50 sequences comprising a STAR. In certain embodiments, the sequences comprising a STAR sequence comprise at least one of the sequences depicted in SEQ ID NOs:1-119.
STAR activity is a feature shared by the sequences listed in SEQ ID NOs:1-119. However, this does not mean that they must all share the same identifier sequence. It is very well possible that different identifiers exist. Identifiers may confer this common feature onto a fragment containing it, though this is not necessarily so.
By using more sequences comprising STAR activity for determining the frequency of occurrence of a sequence pattern or patterns, it is possible to select patterns that are more often than others present or absent in such a STAR sequence. In this way it is possible to find patterns that are very frequently over or under represented in STAR sequences. Frequently over or under represented patterns are more likely to identify candidate STAR sequences in test sets. Another way of using a set of over or under represented patterns is to determine which pattern or combination of patterns is best suited to identify a STAR in a sequence. Using so-called discriminant statistics we have identified a set of patterns which performs best in identifying a sequence comprising a STAR element. In certain embodiments, at least one of the sequence patterns for detecting a STAR sequence comprises a sequence pattern GGACCC (SEQ ID NO:505), CCCTGC (SEQ ID NO:247), AAGCCC (SEQ ID NO:311), CCCCCA (SEQ ID NO:339) and/or AGCACC (SEQ ID NO:377). In certain embodiments, at least one of the sequence patterns for detecting a STAR sequence comprises a sequence pattern CCCN{16}AGC (SEQ ID NO:456), GGCN{9}GAC (SEQ ID NO:577), CACN{13}AGG (SEQ ID NO:802), and/or CTGN{4}GCC (SEQ ID NO:880).
A list of STAR sequences can also be used to determine one or more consensus sequences therein. A consensus sequence for a STAR element is, therefore, also provided herein. This consensus sequence can of course be used to identify candidate STAR elements in a test sequence.
Moreover, once a sequence comprising a STAR element has been identified in a vertebrate it can be used by means of sequence homology to identify sequences comprising a STAR element in other species belonging to a vertebrate. In certain embodiments, a mammalian STAR sequence is used to screen for STAR sequences in other mammalian species. Similarly, once a STAR sequence has been identified in a plant species it can be used to screen for homologous sequences with similar function in other plant species. STAR sequences obtainable by a method as described herein are thus provided. Further provided is a collection of STAR sequences. In certain embodiments, the STAR sequence is a vertebrate or plant STAR sequence. More preferably, the STAR sequence is a mammalian STAR sequence or an angiosperm (monocot, such as rice or dicot, such as Arabidopsis). More preferably, the STAR sequence is a primate and/or human STAR sequence.
A list of sequences comprising STAR activity can be used to determine whether a test sequence comprises a STAR element. There are, as mentioned above, many different methods for using such a list for this purpose. In certain embodiments, a method is provided for determining whether a nucleic acid sequence of about 50-5000 base pairs comprises a STAR sequence, this method comprising: generating a first table of sequence patterns comprising the frequency of occurrence of the patterns in a collection of STAR sequences of the invention, generating a second table of the patterns comprising the frequency of occurrence of the patterns in at least one reference sequence, selecting at least one pattern of which the frequency of occurrence differs between the two tables, determining, within the nucleic acid sequence of about 50-5000 base pairs, the frequency of occurrence of at least one of the selected patterns, and determining whether the occurrence in the test nucleic acid is representative of the occurrence of the selected pattern in the collection of STAR sequences. Alternatively, the determining step comprises determining whether the frequency of occurrence in the test nucleic acid is representative of the frequency of occurrence of the selected pattern in the collection of STAR sequences. In certain embodiments, the method further comprises determining whether the candidate STAR comprises a gene transcription modulating quality using a method described herein. In certain embodiments, the collection of STARs comprises the sequences as depicted in SEQ ID NOs:1-119.
Now multiple methods are disclosed for obtaining a STAR sequence, also provided is an isolated and/or recombinant nucleic acid sequence comprising a STAR sequence by a method as described herein.
A STAR sequence can exert its activity in a directional way, i.e., more to one side of the fragment containing it than to the other. Moreover, STAR activity can be amplified in amount by multiplying the number of STAR elements. The latter suggests that a STAR element may comprise one or more elements comprising STAR activity. Another way of identifying a sequence capable of conferring STAR activity on a fragment containing it comprises selecting from a vertebrate or plant sequence, a sequence comprising STAR activity and identifying whether sequences flanking the selected sequence are conserved in another species. Such conserved flanking sequences are likely to be functional sequences. Such a method for identifying a sequence comprising a STAR element comprising selecting a sequence of about 50 to 5000 base pairs from a vertebrate or plant species comprising a STAR element and identifying whether sequences flanking the selected sequence in the species are conserved in at least one other species. We further provide a method for detecting the presence of a STAR sequence within a nucleic acid sequence of about 50-5000 base pairs, comprising identifying a sequence comprising a STAR sequence in a part of a chromosome of a cell of a species and detecting significant homology between the sequence and a sequence of a chromosome of a different species. The STAR in the different species is thus identified. In certain embodiments, the species comprises a plant or vertebrate species, preferably, a mammalian species. We also provide a method for detecting the presence of a STAR element within a nucleic acid sequence of about 50-5000 base pairs of a vertebrate or plant species, comprising identifying whether a flanking sequence of the nucleic acid sequence is conserved in at least one other species.
It is important to note that methods as disclosed herein for detecting the presence of a sequence comprising a STAR sequence using bioinformatical information are iterative in nature. The more sequences comprising a STAR sequence that are identified with a method as described herein, the more patterns are found to be discriminative between a sequence comprising a STAR sequence and a control sequence. Using these newly found discriminative patterns more sequences comprising a STAR sequence can be identified which in turn enlarge the set of patterns that can discriminate, and so on. This iterative aspect is an important aspect of methods provided herein.
Provided is, amongst others, a method for obtaining a cell which expresses two or more proteins, a method for identifying a cell wherein expression of two or more proteins is in a predetermined ratio and a protein expression unit. In all of these embodiments, the above-described obtainable STAR sequences can be used. For example, a STAR sequence of SEQ ID NOs:1-119, Table 3, Table 4, or Table 5, or combinations thereof. More preferably, the STAR sequence is a vertebrate STAR sequence or a plant STAR sequence. Even more preferably, the vertebrate STAR sequence is a human STAR sequence. It is furthermore preferred to use a STAR sequence from a species from which a gene of interest is expressed. For example, when one would like to express two or more proteins and one of the proteins is a human protein, one preferably includes a human STAR sequence for the expression of the human protein.
As outlined above, the STAR elements flanking an expression unit are the basis of the stable expression of the monomer transgenes over many cell generations. We have demonstrated that STAR elements can protect individual transgenes from silencing. In the invention that capability is extended to more than one expression unit introduced (preferentially) independently in a recombinant host cell. Expression units that are not flanked by STAR elements can undergo significant silencing after only 5-10 culture passages, during which time silencing of the STAR protected units is negligible.
The advantages of a method for obtaining a cell which expresses two or more proteins comprising providing the cell with two or more protein expression units encoding the two or more proteins, characterized in that at least two of the protein expression units comprise at least one STAR sequence, are multifold.
The invention uses STAR sequences for the production of two or more proteins and thereby provided is (1) an increased predictability in the creation of recombinant cell lines that efficiently produce the heterologous multimeric proteins of interest, (2) an increased yield of the heterologous multimeric proteins, (3) stable expression of the heterologous multimeric proteins, even during prolonged cultivation in the absence of selection agent and (4) also provided is favorable transgene expression characteristics without amplification of the transgene. The increased yield of heterologous proteins provided by the invention may be obtained at low transgene copy numbers, without selective co-amplification using, for example, the DHFR/methotrexate system. This results in greater stability, since the transgene copy number is low and is not susceptible to decrease due to recombination (McBurney et al., 2002) or repeat-induced gene silencing (Garrick et al., 1998). Fifth, the broad applicability of the method of the invention includes its utility in a wide range of host cell lines. This is, for example, useful/desirable when a particular multimeric protein is preferably expressed by a particular host cell line (e.g., expression of antibodies from lymphocyte-derived host cell lines).
A method described herein, therefore, provides an improvement of expression of two or more proteins in a cells or host cell.
In certain embodiments, provided is a method for identifying a cell wherein expression of two or more proteins is in a predetermined ratio comprising providing:

    
    
        a collection of cells with two or more protein expression units encoding the two or more proteins,
        selecting cells which express two or more proteins, and
        identifying from the obtained selection, cells that express two or more proteins in the predetermined ratio, characterized in that at least two of the protein expression units comprise at least one STAR sequence.
    
    


The selection of cells which express two or more proteins may, for example, be obtained by performing an SDS-PAGE analysis, a Western blot analysis or an ELISA, which are all techniques which are known by a person skilled in the art and, therefore, need no further elaboration. The identification of cells that express two or more proteins in the predetermined ratio can also be performed by these techniques.
The presence of a STAR sequence in at least two of the protein expression units, again, provide the desired predictability, yield, stability and stoichiometrically balanced availability of the two or more proteins.
Especially when polypeptides of a multimeric protein are produced according to a method of the invention it is desirable to provide the required monomers/subunits in a ratio that is relevant for the formation of the multimeric protein. Hence, preferably, the monomers/subunits are produced in a biological relevant balanced ratio. If, for example, a multimeric protein consists of two subunits A and one subunit B, it is desired to produce two subunits A for every subunit of B that is produced. Hence, a predetermined ratio is herein defined as the natural occurring ratio (stoichiometry) of the different subunits/monomers/polypeptides which comprise a multimeric protein.
In a more preferred embodiment, a cell obtainable according to a method of the invention expresses two proteins. For example, two proteins which together provide a therapeutically advantageous effect. In an even more preferred embodiment, the predetermined ratio of the two expressed proteins is 1:1. This is, for example, useful in the production of multimeric proteins in which the monomers are in a 1:1 ratio. Typical examples are antibodies that comprise two heavy chains and two light chains.
Preferably, provided is a method, wherein two or more protein expression units further encode at least two different selection markers, and wherein the method further comprises a two-step selection marker screening on the cell, wherein the cell is selected in a first step on the presence of a first selection marker and in a second step on the presence of a second selection marker.
In this embodiment of the invention, a two-stage antibiotic selection regime is used which results in a high proportion of isolates that express, for example, transgenes 1 and 2 at high levels; the first stage of selection eliminates cells that do not contain the expression unit or units, and the second stage of selection eliminates colonies that do not transcribe both bicistronic mRNAs at high levels. This regime is one of the aspects for the increased frequency of multimer-expressing recombinant cell lines achieved by the invention compared to conventional methods. As described herein, it results in an increase in the frequency of expressor lines by more than ten-fold.
In certain embodiments, provided is a method wherein at least one of the protein expression units comprises a monocistronic gene comprising an open reading frame encoding a protein of interest and wherein the monocistronic gene is under control of a functional promoter.
In certain embodiments, provided is a method, wherein at least one of the protein expression units comprises: a bicistronic gene comprising an open reading frame encoding a protein of interest, a protein translation initiation site with a reduced translation efficiency, and a selection marker, wherein the bicistronic gene is under control of a functional promoter.
In a more preferred embodiment, provided is a method, wherein at least one of the protein expression units comprises: a bicistronic gene comprising an open reading frame encoding a protein of interest, a protein translation initiation site with a reduced translation efficiency, and a selection marker, wherein the bicistronic gene is under control of a functional promoter, which protein expression unit further comprises: a monocistronic gene comprising an open reading frame encoding a second selection marker, wherein the monocistronic gene is under control of a functional promoter.



DESCRIPTION OF THE FIGURES
The drawings show representative versions of the DNA molecules of the invention. These portions of DNA, referred to as (a) protein expression unit(s), is/are created and manipulated in vectors such as recombinant plasmid molecules and/or recombinant viral genomes. The protein expression units are integrated into host cell genomes as part of the method of the invention, and the schematic drawings represent the configuration of the DNA elements in the expression units in both the vector molecules and the host cell genome.
FIG. 1A is a schematic diagram that shows the first expression unit. It is flanked by STAR elements, and comprises a bicistronic gene containing (from 5′ to 3′) a transgene (encoding, for example, a reporter gene or one subunit of a multimeric protein; TG S1, “transgene subunit 1”), an IRES, and a selectable marker (zeo, conferring zeocin resistance) under control of the CMV promoter. A monocistronic selectable marker (neo, conferring G418 resistance) under control of the SV40 promoter is included. Both genes have the SV40 transcriptional terminator at their 3′ ends (t).
FIG. 1B is a schematic diagram that shows the second expression unit. It is flanked by STAR elements, and contains a bicistronic gene containing (from 5′ to 3′) a transgene (encoding, for example, a different reporter gene or another subunit of a multimeric protein; TG S2), an IRES, and a selectable marker (bsd, conferring blasticidin resistance) under control of the CMV promoter. A monocistronic selectable marker (neo, conferring G418 resistance) under control of the SV40 promoter is included. Both genes have the SV40 transcriptional terminator at their 3′ ends.
FIG. 2 is a diagram of the pSDH-CSP plasmid. The Secreted Alkaline Phosphatase (SEAP) reporter gene is under control of the CMV promoter, and the puromycin resistance selectable marker (puro) is under control of the SV40 promoter. Flanking these two genes are multiple cloning sites into which STAR elements can be cloned. The plasmid also has an origin of replication (ori) and ampicillin resistance gene (ampR) for propagation in Escherichia coli. 
FIGS. 3A and 3B are diagrams of the pSDH-SIB/Z and pSDH-GIB/Z families of plasmids. These plasmids are derived from the pSDH-CSP plasmid (FIG. 2), by replacement of the monocistronic SEAP and puro genes with a bicistronic gene under control of the CMV promoter and a monocistronic neomycin resistance selectable marker gene (neo) under control of the SV40 promoter. FIG. 3A depicts pSDH-SIB/Z in which the bicistronic gene encodes secreted alkaline phosphatase (SEAP) in the 5′ position and blasticidin (bsd) or zeocin (zeo) resistance selectable markers in the 3′ position, relative to the IRES. FIG. 3B illustrates pSDH-GIB/Z in which the bicistronic gene encodes green fluorescent protein (GFP) in the 5′ position and blasticidin (bsd) or zeocin (zeo) resistance selectable markers in the 3′ position, relative to the IRES.
FIG. 4 is a comparison of the consequences of one-step and two-step antibiotic selection on the predictability of transgene expression. Recombinant CHO cell isolates containing plasmid pSDH-SIZ or plasmid pSDH-SIZ-STAR18 were selected on G418 (panel A) or sequentially on G418 and zeocin (panel B) and assayed for SEAP activity.
FIG. 5 is a schematic diagram illustrating the PP (Plug and Play) family of plasmids. These plasmids contain a bicistronic expression unit (containing an IRES) between multiple cloning sites (MCS) for insertion of STAR elements. MCSI, SbfI-SalI-XbaI-AscI-SwaI; MCSII, BsiWI-EcoRV-BglII-PacI. Panel A, the bicistronic gene encodes GFP and the puromycin resistance marker (puro). Panel B, the bicistronic gene encodes secreted alkaline phosphatase (SEAP) and the zeocin resistance marker (zeo). Panel C, the bicistronic gene encodes SEAP and the neocin resistance marker (neo). Panel D, the bicistronic gene encodes GFP and puro, and an adjacent monocistronic gene encodes neo. Panel E, the bicistronic gene encodes SEAP and zeo, and an adjacent monocistronic gene encodes neo. Bicistronic genes are under control of the CMV promoter (pCMV) and the monocistronic gene is under control of the SV40 promoter (pSV40). A stuffer fragment of 0.37 kb (St) separates MCSI from pCMV. Both the bicistronic and monocistronic genes have the SV40 polyadenylation site at their 3′ ends.
FIG. 6 is a diagram depicting the pSDH-CSP plasmid used for testing STAR activity. The Secreted Alkaline Phosphatase (SEAP) reporter gene is under control of the CMV promoter, and the puromycin resistance selectable marker (puro) is under control of the SV40 promoter. Flanking these two genes are multiple cloning sites into which STAR elements can be cloned. The plasmid also has an origin of replication (ori) and ampicillin resistance gene (ampR) for propagation in Escherichia coli. 
FIG. 7 is a graph illustrating that STAR6 (SEQ ID NO:6) and STAR49 (SEQ ID NO:49) improve predictability and yield of transgene expression. Expression of SEAP from the CMV promoter by CHO cells transfected with pSDH-CSP, pSDH-CSP-STAR6, or pSDH-CSP-STAR49 was determined. The STAR-containing constructs confer greater predictability and elevated yield relative to the pSDH-CSP construct alone.
FIG. 8 is a graph illustrating that STAR6 (SEQ ID NO:6) and STAR8 (SEQ ID NO:8) improve predictability and yield of transgene expression. Expression of luciferase from the CMV promoter by U-2 OS cells transfected with pSDH-CMV, pSDH-CMV-STAR6, or pSDH-CMV-STAR8 was determined. The STAR-containing constructs confer greater predictability and elevated yield relative to the pSDH-CMV construct alone.
FIG. 9 is a graph showing the minimal essential sequences of STAR10 (SEQ ID NO:10) and STAR27 (SEQ ID NO:27). Portions of the STAR elements were amplified by PCR: STAR10 (SEQ ID NO:10) was amplified with primers E23 (SEQ ID NO:172) and E12 (SEQ ID NO:161) to yield fragment 10A, E13 (SEQ ID NO:162) and E14 (SEQ ID NO:163) to yield fragment 10B, and E15 (SEQ ID NO:164) and E16 (SEQ ID NO:165) to yield fragment 10C. STAR27 (SEQ ID NO:27) was amplified with primers E17 (SEQ ID NO:166) and E18 (SEQ ID NO:167) to yield fragment 27A, E19 (SEQ ID NO:168) and E20 (SEQ ID NO:169) to yield fragment 27B, and E21 (SEQ ID NO:170) and E22 (SEQ ID NO:171) to yield fragment 27C. These sub-fragments were cloned into the pSelect vector. After transfection into U-2 OS/Tet-Off/LexA-HP1 cells, the growth of the cultures in the presence of zeocin was monitored. Growth rates varied from vigorous (+++) to poor (+/−), while some cultures failed to survive zeocin treatment (−) due to absence of STAR activity in the DNA fragment tested.
FIG. 10 is a graph showing STAR element function in the context of the SV40 promoter. pSDH-SV40 and pSDH-SV40-STAR6 were transfected into the human osteosarcoma U-2 OS cell line, and expression of luciferase was assayed with or without protection from gene silencing by STAR6 (SEQ ID NO:6) in puromycin-resistant clones.
FIG. 11 is a graph illustrating STAR element function in the context of the Tet-Off promoter. pSDH-Tet and pSDH-Tet-STAR6 were transfected into the human osteosarcoma U-2 OS cell line, and expression of luciferase was assayed with or without protection from gene silencing by STAR6 (SEQ ID NO:6) in puromycin-resistant clones.
FIG. 12 is a schematic diagram illustrating the orientation of STAR elements as they are cloned in the pSelect vector (panel A), as they are cloned into pSDH vectors to preserve their native orientation (panel B), and as they are cloned into pSDH vector in the opposite orientation (panel C).
FIG. 13 is a graph showing directionality of STAR66 (SEQ ID NO:66) function. The STAR66 element (SEQ ID NO:66) was cloned into pSDH-Tet in either the native (STAR66 native) or the opposite orientation (STAR66 opposite), and transfected into U-2 OS cells. Luciferase activity was assayed in puromycin resistant clones.
FIG. 14 is a southern blot showing copy number-dependence of STAR function. Southern blot of luciferase expression units in pSDH-Tet-STAR10, integrated into U-2 OS genomic DNA. Radioactive luciferase DNA probe was used to detect the amount of transgene DNA in the genome of each clone, which was then quantified with a phosphorimager.
FIG. 15 is a graph illustrating copy number-dependence of STAR function. The copy number of pSDH-Tet-STAR10 expression units in each clone was determined by phosphorimagery, and compared with the activity of the luciferase reporter enzyme expressed by each clone.
FIG. 16 contains graphs illustrating enhancer-blocking and enhancer assays. The luciferase expression vectors used for testing STARs for enhancer-blocking and enhancer activity are shown schematically. The E-box binding site for the E47 enhancer protein is upstream of a cloning site for STAR elements. Downstream of the STAR cloning site is the luciferase gene under control of a human alkaline phosphatase minimal promoter (mp). The histograms indicate the expected outcomes for the three possible experimental situations (see text). Panel A: Enhancer-blocking assay. Panel B: Enhancer assay.
FIG. 17 is a graph depicting the enhancer-blocking assay. Luciferase expression from a minimal promoter is activated by the E47/E-box enhancer in the empty vector (vector). Insertion of enhancer-blockers (scs, HS4) or STAR elements (STAR elements 1, 2, 3, 6, 10, 11, 18, and 27; SEQ ID NOS:1, 2, 3, 6, 10, 11, 18, and 27, respectively) block luciferase activation by the E47/E-box enhancer.
FIG. 18 is a graph illustrating an enhancer assay. Luciferase expression from a minimal promoter is activated by the E47/E-box enhancer in the empty vector (E47). Insertion of the scs and HS4 elements or various STAR elements (STARs 1, 2, 3, 6, 10, 11, 18, and 27; SEQ ID NOS:1, 2, 3, 6, 10, 11, 18, and 27, respectively) do not activate transcription of the reporter gene.
FIG. 19 contains graphs illustrating STAR18 (SEQ ID NO:18) sequence conservation between mouse and human. The region of the human genome containing 497 base pair STAR18 (SEQ ID NO:18) is shown (black boxes); the element occurs between the HOXD8 and HOXD4 homeobox genes on human chromosome 2. It is aligned with a region in mouse chromosome 2 that shares 72% sequence identity. The region of human chromosome 2 immediately to the left of STAR18 is also highly conserved with mouse chromosome 2 (73% identity; gray boxes); beyond these region, the identity drops below 60%. The ability of these regions from human and mouse, either separately or in combination, to confer growth on zeocin is indicated: −, no growth; +, moderate growth; ++, vigorous growth; +++, rapid growth.
FIG. 20 is a schematic diagram of bio-informatical analysis workflow. For details, see text.
FIG. 21 is a diagram illustrating the results of discriminant analysis on classification of the training set of 65 STAR elements. STAR elements that are correctly classified as STARs by Stepwise Linear Discriminant Analysis (LDA) are shown in a Venn diagram. The variables for LDA were selected from frequency analysis results for hexameric oligonucleotides (“oligos”) and for dyads. The diagram indicates the concordance of the two sets of variables in correctly classifying STARs.
FIG. 22 is a graph depicting the RT-PCR assay of Arabidopsis STAR strength. U-2 OS/Tet-Off/lexA-HP1 cells were transfected with candidate Arabidopsis STAR elements and cultivated at low doxycycline concentrations. Total RNA was isolated and subjected to RT-PCR; the bands corresponding to the zeocin and hygromycin resistance mRNAs were detected by Southern blotting and quantified with a phosphorimager. The ratio of the zeocin to hygromycin signals is shown for transfectants containing zeocin expression units flanked by 12 different Arabidopsis STAR elements, the Drosophila scs element, or no flanking element.
FIG. 23 includes schematic diagrams and a graph illustrating that STAR elements allow efficient and simultaneous expression of two genes from two distant vectors. The ppGIZ, ppGIZ-STAR7, ppRIP and ppRIP-STAR7 vectors used for testing simultaneous expression of respectively GFP and RED are shown. The expression unit comprises (from 5′ to 3′) genes encoding the GFP or RED proteins, an IRES, and a selectable marker (zeo, conferring zeocin resistance or respectively puro, puromycin resistance gene) under control of the CMV promoter. The expression unit has the SV40 transcriptional terminator at its 3′ end (t). The cassettes with the GFP and RED expression units are either flanked by STAR7 elements (SEQ ID NO:7) (STAR7-shielded) or not (Control). The two control constructs or the two STAR7-shielded vectors are simultaneously transfected to CHO-K1 cells. Stable colonies that are resistant to both zeocin and puromycin are expanded and the GFP and RED signals are determined on a XL-MCL Beckman Coulter flow cytometer. The percentage of cells in one colony that are double positive for both GFP and RED signals is taken as measure for simultaneous expression of both proteins and this is plotted in FIG. 23.
FIG. 24 also includes schematic diagrams and a graph illustrating that STAR elements improve expression of a functional antibody in CHO cells. The different vectors containing the Light and Heavy Chain of the RING1 antibody are shown in FIG. 24. The constructs are simultaneously transfected to CHO cells. Stable colonies that are resistant to both zeocin and puromycin are expanded. The cell culture medium of these colonies is tested for the detection of functional RING1 antibody in an ELISA with RING1 protein as antigen. The values are dividing by the number of cells in the colony. The highest value detected in the STAR-less control is arbitrarily set at 100%.



DETAILED DESCRIPTION
Definitions
Cell, Host Cell, Cell Line, Host Cell Line
The terms “cell”/“host cell” and “cell line”/“host cell line” are respectively typically defined as a eukaryotic cell and homogeneous populations thereof that are maintained in cell culture by methods known in the art, and that have the ability to express heterologous proteins.
Expression
The term “expression” is typically used to refer to the production of a specific RNA product or products, or a specific protein or proteins, in a cell. In the case of RNA products, it refers to the process of transcription. In the case of protein products, it refers to the processes of transcription, translation and optionally post-translational modifications. In the case of secreted proteins, it refers to the processes of transcription, translation, and optionally post-translational modification (e.g., glycosylation, disfulfide bond formation, etc.), followed by secretion. In the case of multimeric proteins, it includes assembly of the multimeric structure from the polypeptide monomers. The corresponding verbs of the noun “expression” have an analogous meaning as the noun.
Protein, Multimer, Multimeric Protein
A protein is herein defined as being either (i) a product obtained by the processes of transcription and translation and possibly but not necessarily a product that is part of a multimeric protein (for example, a subunit) and/or (ii) a product obtained by the processes of transcription, translation and post-translational modification. The term “multimer” or “multimeric protein” is typically defined as a protein that comprises two or more, possibly non-identical, polypeptide chains (“monomers”). The different monomers in a multimeric protein can be present in stoichiometrically equal or unequal numbers. In either case, the proportion of the monomers is usually fixed by the functional structure of the multimeric protein.
Protein Expression Unit
The term “protein expression unit” is herein defined as a unit capable of providing protein expression and typically comprises a functional promoter, an open reading frame encoding a protein of interest and a functional terminator, all in operable configuration. A functional promoter is a promoter that is capable of initiating transcription in a particular cell. Suitable promoters for obtaining expression in eukaryotic cells are the CMV-promoter, a mammalian EF1-alpha promoter, a mammalian ubiquitin promoter, or a SV40 promoter. A functional terminator is a terminator that is capable of providing transcription termination. One example of a suitable terminator is an SV40 terminator.
An Open Reading Frame Encoding a Protein of Interest (or a Transgene)
The term “an open reading frame encoding a protein of interest (or a transgene)” is typically defined as a fragment of DNA which codes for a specific RNA product or products or a specific protein or proteins, and which is optionally capable of becoming integrated into the genome of a host cell. It includes DNA elements required for proper transcription and translation of the coding region(s) of the transgene. The DNA encoding the protein of interest/transgene can either be a DNA encoding a product obtained by the processes of transcription and translation (and possibly but not necessarily this product is part of a multimeric protein, for example, a subunit) or a product obtained by the processes of transcription, translation and post-translational modification.
Recombinant Cell, Recombinant Host Cell, Recombinant Cell Line, Recombinant Host Cell Line
The terms “recombinant cell/host cell” and “recombinant cell line/host cell line” are respectively typically defined as a host cell and homogeneous populations thereof into which a transgene has been introduced for the purpose of producing a heterologous protein or proteins.
STAR (Stabilizing Anti-Repressor) Sequence & STAR Element
A STAR (STabilizing Anti-Repressor) sequence (or STAR element; these terms will be used interchangeably herein) is a naturally occurring DNA element that we have isolated from eukaryotic genomes on the basis of their ability to block transgene repression. In certain embodiments, the STAR elements are recovered from the human genome. A STAR sequence comprises the capacity to influence transcription of genes in cis and/or provide a stabilizing and/or an enhancing effect. It has been demonstrated that when STAR elements flank transgenes, the transgene expression level of randomly selected recombinant cell lines can be increased to levels approaching the maximum potential expression of the transgene's promoter. Moreover, the expression level of the transgene is stable over many cell generations, and does not manifest stochastic silencing. Therefore, STAR sequences confer a degree of position-independent expression on transgenes that is not possible with conventional transgenic systems. The position independence means that transgenes that are integrated in genomic locations that would result in transgene silencing are, with the protection of STAR elements, maintained in a transcriptionally active state.
Quality
The term quality in relation to a sequence refers to an activity of the sequence.
STAR, STAR Sequence, STAR Element
The term STAR, STAR sequence or STAR element, as used herein, refers to a DNA sequence comprising one or more of the mentioned gene transcription modulating qualities.
DNA Sequence
The term “DNA sequence” as used herein does, unless otherwise specified, not refer to a listing of specific ordering of bases but rather to a physical piece of DNA. A transcription quality with reference to a DNA sequence refers to an effect that the DNA sequence has on transcription of a gene of interest. “Quality” as used herein refers to detectable properties or attributes of a nucleic acid or protein in a transcription system.
Bicistronic Gene
The term “bicistronic gene,” is typically defined as a gene capable of providing a RNA molecule that encodes two proteins/polypeptides.
Monocistronic Gene
The term “monocistronic gene” is typically defined as a gene capable of providing a RNA molecule that encodes one protein/polypeptide.
Selection Marker or Selectable Marker
The term “selection marker or selectable marker” is typically used to refer to a gene and/or protein whose presence can be detected directly or indirectly in a cell, for example, a gene and/or a protein that inactivates a selection agent and protects the host cell from the agent's lethal or growth-inhibitory effects (e.g., an antibiotic resistance gene and/or protein). Another possibility is that the selection marker induces fluorescence or a color deposit (e.g., green fluorescent protein and derivatives, luciferase, or alkaline phosphatase).
Selection Agent
The term “selection agent” is typically defined as a chemical compound that is able to kill or retard the growth of host cells (e.g., an antibiotic).
Selection
The term “selection” is typically defined as the process of using a selection marker/selectable marker and a selection agent to identify host cells with specific genetic properties (e.g., that the host cell contains a transgene integrated into its genome).
Clone, Isolate
The nouns “clone” and “isolate” typically refer to a recombinant host cell line that has been identified and isolated by means of selection.
The improvements provided by a method described herein have three integrated aspects. (1) With existing systems, recombinant cell lines that simultaneously express acceptable quantities of the monomers of multimeric proteins can be created only at very low frequencies; the invention increases the predictability of creating high-yielding recombinant host cell lines by a factor of ten or more. (2) Existing systems do not provide stoichiometrically balanced and proportional amounts of the subunits of multimeric proteins; the invention ensures that the expression levels of the subunits will be balanced and proportional. (3) Existing systems do not provide a means of protecting the transgenes that encode the protein subunits from transgene silencing.
FIG. 1 provides a non-limiting schematic representation of one of the embodiments of this part of the invention. FIGS. 1A and 1B show two separate protein expression units. This is the configuration of the DNA elements of the expression units in the plasmid as well as after integration into the genome. Expression unit one is shown in FIG. 1A. It contains an open reading frame for a transgene (a reporter gene or subunit 1 of a multimeric (TG S1, transgene subunit 1)). This is upstream of the attenuated EMCV IRES, and of the open reading frame encoding the zeocin resistance selectable marker protein (zeo). This bicistronic transgene is transcribed at high levels from the CMV promoter. Next to this is the neomycin resistance selectable marker (neo; this confers resistance to the antibiotic G418 as well), transcribed as a monocistronic mRNA from the SV40 promoter. These two genes are flanked by STAR elements. In FIG. 1B a similar expression unit is depicted. It consists of a second transgene (a second reporter gene or the open reading frame for subunit 2 of a heterodimeric protein (TG S2)) upstream of the attenuated EMCV IRES and the blasticidin selectable marker open reading frame (bsd). This bicistronic transgene is transcribed at high levels from the CMV promoter. Next to this is the neo selectable marker, transcribed as a monocistronic mRNA from the SV40 promoter. The two genes in the second expression unit are flanked by STAR elements as well.
The possible combinations of selection markers are numerous. Examples of possible antibiotic combinations are provided above. The one antibiotic that is particularly advantageous is zeocin, because the zeocin-resistance protein (zeocin-R) acts by binding the drug and rendering it harmless. Therefore, it is easy to titrate the amount of drug that kills cells with low levels of zeocin-R expression, while allowing the high-expressors to survive. All other antibiotic-resistance proteins in common use are enzymes, and thus act catalytically (not 1:1 with the drug).
When a two-step selection is performed it is, therefore, advantageous to use an antibiotic resistance protein with this 1:1 binding mode of action. Hence, the antibiotic zeocin is a preferred selection marker. For convenience the zeocin antibiotic is in a two-step selection method combined with puromycin-R or blasticidin-R in the second bicistronic gene, and neomycin-R or hygromycin-R in the monocistronic gene.
It is also possible to combine an antibiotic selection marker with a selection marker which provides induction of fluorescence or which provides a color deposit.
Different promoters can be used as long as they are functional in the used cell. The CMV promoter is considered the strongest available, so it is preferably chosen for the bicistronic gene in order to obtain the highest possible product yield. Other examples of suitable promoters are, e.g., mammalian promoters for EF1-alpha or ubiquitin. The good expression and stability of the SV40 promoter makes it well suited for expression of the monocistronic gene; enough selection marker protein (for example, the antibiotic resistance protein neomycin-R in the example cited herein) is made to confer high expression of the selection marker. Hence, the SV40 promoter is preferentially used as a promoter driving the expression of the selection marker.
In certain embodiments, provided is a method wherein at least one of the protein expression units comprises at least two STAR sequences. In an even more preferred embodiment, provided is a method wherein the protein expression unit comprising at least two STAR sequences is arranged such that the protein expression unit is flanked on either side by at least one STAR sequence. In yet an even more preferred embodiment, the at least two STAR sequences are essentially identical. Essentially identical STAR sequences are defined herein as STAR sequences which are identical in their important domains, but which may vary within their less important domains (the domains that confer the transcription stabilizing or enhancing quality), for example, a point mutation, deletion or insertion at a less important position within the STAR sequence. Preferentially, the essentially identical STAR sequences provide equal amounts of transcription stabilizing or enhancing activity.
The use of STAR sequences to flank at least one protein expression unit is one of the aspects of the balanced and proportional levels of expression of two or more proteins and more specifically for the expression of the monomers of multimeric proteins. The STAR sequences create chromatin domains of definite and stable transcriptional potential. As a result, promoters that drive transcription of each bicistronic mRNA will function at definite, stable levels. A recombinant host cell line created by the method of the invention is readily identified in which these levels result in appropriate proportions of each monomer of the multimeric protein of interest being expressed at high yields.
In certain embodiments, the protein expression unit contains only the bicistronic gene flanked by STAR elements. The advantages of omitting the monocistronic antibiotic resistance gene are twofold. First, selection of high-expressing recombinant host cells requires the use of only two antibiotics. Second, it prevents repression of the bicistronic and/or monocistronic genes by the phenomena of promoter suppression and transcriptional interference. These phenomena are common problems in conventional transgenic systems in which two or more transcription units are located near each other. Repression by an upstream (5′) unit of a downstream (3′) unit is termed transcriptional interference, and repression by a downstream unit of an upstream unit is termed promoter suppression (Villemure et al., 2001). Transcriptional interference can result in suppression of adjacent transgenes in all possible arrangements (tandem, divergent, and convergent) (Eszterhas et al., 2002). These phenomena can reduce the efficiency of selection of the IRES-dependent and/or monocistronic antibiotic resistance genes, and reduce the yield of the transgene. Therefore, the embodiment of the invention comprising only a bicistronic gene flanked by STAR elements provides an alternative configuration of the components.
In certain embodiments, the method described herein uses a STAR sequence wherein the STAR sequence is depicted in Table 3 and/or SEQ ID NOs:1-119 and/or a functional equivalent and/or a functional fragment thereof.
We have isolated and characterized an extensive collection of STAR sequences using proprietary technology. The strength of these sequences ranges widely. This is manifested by the varying degrees of improvement of transgene expression in recombinant host cells conferred by the STAR elements; some STAR elements provide full protection from silencing, while others only provide partial protection. The range in strength of the STAR elements is also manifested in their varying capacities to improve the predictability of isolating recombinant cell lines that efficiently produce the heterologous proteins of interest. For the invention we have preferably employed STAR elements that have strong predictability characteristics, in order to have high numbers of efficiently-expressing recombinant cell lines. The STAR elements employed have moderate to strong anti-repressor activity, in order to be able to modulate the levels of recombinant protein production to match the requirements of the product (e.g., balanced and proportional expression of polypeptide monomers). The selected STAR elements also confer significant increases on the stability of expression of the transgenes.
Some STAR elements also display promoter and host cell-type specificity. These characteristics are exploited to create novel transgenic systems to optimize the production of heterologous proteins that require a specific host cell (for example, to achieve a high yield or a pharmaceutically advantageous glycosylation pattern) or a specific mode of expression (for example, the use of an inducible promoter or a constitutive promoter; the use of a promoter with moderate strength or high strength, etc.). Therefore, the use of different STAR elements results in different embodiments of the invention that pertain to these types of applications.
A functional equivalent and/or a functional fragment of a sequence depicted in Table 3 and/or SEQ ID NOs:1-119 is defined herein as follows. A functional equivalent of a sequence as depicted in Table 3 and/or SEQ ID NOs:1-119 is a sequence derived with the information given in Table 3 and/or SEQ ID NOs:1-119. For instance, a sequence that can be derived from a sequence in Table 3 and/or SEQ ID NOs:1-119 by deleting, modifying and/or inserting bases in or from a sequence listed in Table 3 and/or SEQ ID NOs:1-119, wherein the derived sequence comprises the same activity in kind, not necessarily in amount, of a sequence as depicted in Table 3 and/or SEQ ID NOs:1-119. A functional equivalent is further a sequence comprising a part from two or more sequences depicted in Table 3 and/or SEQ ID NOs:1-119. A functional equivalent can also be a synthetic DNA sequence which is a sequence that is not derived directly or indirectly from a sequence present in an organism. For instance, a sequence comprising a drosophila scs or scs' sequence is not a synthetic sequence, even when the scs or scs' sequence was artificially generated.
Functional sequences of STAR elements can be delineated by various methods known in the art. In certain embodiments, deletions and/or substitutions are made in STAR sequences. DNA that is modified in such a way is, for example, tested for activity by using a single modified nucleic acid or by generating a collection of test nucleic acids comprising the modified nucleic acid. Elucidation of functional sequences within STAR sequences enables the elucidation of consensus sequences for elements with a gene transcription modulating and/or a gene transcription repressing quality.
A functional fragment of a STAR sequence as depicted in Table 3 and/or SEQ ID NOs:1-119 can, for example, be obtained by deletions from the 5′ end or the 3′ end or from the inside of the sequences or any combination thereof, wherein the derived sequence comprises the same activity in kind, not necessarily in amount.
In a more preferred embodiment, the STAR sequence as depicted in Table 3 and/or SEQ ID NOs:1-119 is STAR18 and/or a functional equivalent and/or a functional fragment thereof.
Yet another preferred feature of a method described herein is the introduction of a (weak) IRES as an example of a protein translation initiation site with a reduced translation efficiency, between the open reading frame of the protein of interest and the selection marker open reading frame. In combination with, for example, the STAR sequence, this component of the invention comprises a marked improvement in transgenic systems for the expression of two or more proteins.
IRES elements are known from viral and mammalian genes (Martinez-Salas, 1999), and have also been identified in screens of small synthetic oligonucleotides (Venkatesan & Dasgupta, 2001). The IRES from the encephalomyocarditis virus has been analyzed in detail (Mizuguchi et al., 2000). An IRES is an element encoded in DNA that results in a structure in the transcribed RNA at which eukaryotic ribosomes can bind and initiate translation. An IRES permits two or more proteins to be produced from a single RNA molecule (the first protein is translated by ribosomes that bind the RNA at the cap structure of its 5′ terminus, (Martinez-Salas, 1999)). Translation of proteins from IRES elements is less efficient than cap-dependent translation: the amount of protein from IRES-dependent open reading frames (ORFs) ranges from less than 20% to 50% of the amount from the first ORF (Mizuguchi et al., 2000). This renders IRES elements undesirable for production of all subunits of a multimeric protein from one messenger RNA (mRNA), since it is not possible to achieve balanced and proportional expression of two or more protein monomers from a bicistronic or multicistronic mRNA. However, the reduced efficiency of IRES-dependent translation provides an advantage that is exploited by the current invention. Furthermore, mutation of IRES elements can attenuate their activity, and lower the expression from the IRES-dependent ORFs to below 10% of the first ORF (Lopez de Quinto & Martinez-Salas, 1998, Rees et al., 1996). The advantage exploited by the invention is as follows: when the IRES-dependent ORF encodes a selectable marker protein, its low relative level of translation means that high absolute levels of transcription must occur in order for the recombinant host cell to be selected. Therefore, selected recombinant host cell isolates will by necessity express high amounts of the transgene mRNA. Since the recombinant protein is translated from the cap-dependent ORF, it can be produced in abundance resulting in high product yields.
Changes to the IRES can be made without altering the essence of the function of the IRES (hence, providing a protein translation initiation site with a reduced translation efficiency), resulting in a modified IRES. Use of a modified IRES which is still capable of providing a small percentage of translation (compared to a 5′ cap translation) is, therefore, also included in this invention.
In certain embodiments, provided is a method for obtaining a cell which expresses two or more proteins or a method for identifying a cell wherein expression of two or more proteins is in a predetermined ratio, wherein each of the protein expression units resides on a separate DNA-carrier. The invention preferentially makes use of a separate transcription unit for each protein and/or monomer of a multimeric protein. In each transcription unit the monomer ORF is produced by efficient cap-dependent translation. This feature of provided is isolated recombinant host cells that have high yields of each monomer, at levels that are balanced and proportionate to the stoichiometry of the multimeric protein. The increased predictability at which such recombinant host cells are isolated results in an improvement in the efficiency of screening for such isolates by a factor of ten or more. In certain embodiments, the DNA-carrier is a vector (or plasmid; the terms are used interchangeably herein). In certain embodiments, the vector is a viral vector and in a more preferred embodiment, the viral vector is an adenoviral vector or a retroviral vector. Other viral vectors can also be used in a method described herein.
Conventional expression systems are DNA molecules in the form of a recombinant plasmid or a recombinant viral genome. The plasmid or the viral genome is introduced into (mammalian host) cells and integrated into their genomes by methods known in the art. The invention also uses these types of DNA molecules to deliver its improved transgene expression system. A preferred embodiment of the invention is the use of plasmid DNA for delivery of the expression system. A plasmid contains a number of components: conventional components, known in the art, are an origin of replication and a selectable marker for propagation of the plasmid in bacterial cells; a selectable marker that functions in eukaryotic cells to identify and isolate host cells that carry an integrated transgene expression system; the protein of interest, whose high-level transcription is brought about by a promoter that is functional in eukaryotic cells (e.g., the human cytomegalovirus major immediate early promoter/enhancer, pCMV (Boshart et al., 1985)); and viral transcriptional terminators for the transgene of interest and the selectable marker (e.g., the SV40 polyadenylation site (Kaufman & Sharp, 1982)).
The vector used can be any vector that is suitable for cloning DNA and that can be used in a transcription system. When host cells are used it is preferred that the vector is an episomally replicating vector. In this way, effects due to different sites of integration of the vector are avoided. DNA elements flanking the vector at the site of integration can have effects on the level of transcription of the promoter and thereby mimic effects of fragments comprising DNA sequences with a gene transcription modulating quality. In certain embodiments, the vector comprises a replication origin from the Epstein-Barr virus (EBV), OriP, and a nuclear antigen (EBNA-1). Such vectors are capable of replicating in many types of eukaryotic cells and assemble into chromatin under appropriate conditions.
In certain embodiments, provided is a method for obtaining a cell which expresses two or more proteins or a method for obtaining a cell wherein expression of two or more proteins is in a predetermined ratio comprising providing two or more protein expression units wherein one of the protein expression units or protein(s) of interest encodes an immunoglobulin heavy chain and/or wherein another of the protein expression units or protein(s) of interest encodes an immunoglobulin light chain. According to this embodiment, a multimeric protein, an antibody, is obtained. It is possible to provide a cell which expresses an immunoglobulin heavy chain from one protein expression unit and an immunoglobulin light chain from another protein expression unit with a third protein expression unit encoding a secretory component or a joining chain. In this way the production of, for example, sIgA and pentameric IgM is provided.
Preferably, the used host cell secretes the produced multimer. In this way, the product is easily isolated from the medium surrounding the host cell.
More preferably, the invention results in the production of a functional multimer. The functionality of the produced multimer is determined with standard procedures. For example, a produced multi subunit enzyme is tested in a corresponding enzymatic assay or by binding to an antigen, for example, in an ELISA used to test the functionality of a produced antibody.
Hence, the selection of a final suitable host cell expressing a multimer involves multiple steps amongst which are the selection for a cell that expresses all the desired subunits of a multimer, followed by a functional analysis of the multimer.
With regard to a multimeric protein, high expression levels of the subunits is desired as well as the formation of a functional multimeric protein of the subunits. Surprisingly, the use of a STAR sequence for the production of the subunits of a multimeric protein results in a high amount of cells that express the subunits, as compared to control vectors without a STAR sequence. Moreover, the amount of functional multimeric protein is relatively higher when compared to the control.
Production of subunits and the formation of functional multimeric protein from these subunits is in particular of importance for the production of antibodies. When the heavy chain and light chain expression cassette are flanked by a STAR sequence this results in a higher production of functional antibody, as compared to control vectors without a STAR sequence. Hence, the presence of a STAR sequence results in a higher degree of predictability of functional antibody expression. In certain embodiments, each expression unit comprises at least two STAR sequences which sequences are arranged such that the expression unit is flanked on either side by at least one STAR sequence.
In certain embodiments, a method is provided, wherein the protein expression units are introduced simultaneously into the cell.
Preferably, a functional promoter is a human cytomegalovirus (CMV) promoter, a simian virus (SV40) promoter, a human ubiquitin C promoter or a human elongation factor alpha (EF1-α) promoter.
As disclosed herein within the experimental part, a STAR sequence can confer copy number-dependence on a transgene expression unit, making transgene expression independent of other transgene copies in tandem arrays, and independent of gene-silencing influences at the site of integration. Hence, also provided is a method for obtaining a cell which expresses two or more proteins or a method for identifying a cell wherein expression of two or more proteins is in a predetermined ratio in which multiple copies of a protein expression unit encoding a protein of interest is integrated into the genome of the cell (i.e., in which cell, an amplification of the gene of interest is present).
According to this part of the invention, the protein expression units are introduced simultaneously into the cells or host cell or collection of cells by methods known in the art. Recombinant host cells are selected by treatment with an appropriate antibiotic, for example, G418, using methods known in the art. After formation of individual antibiotic-resistant colonies, another antibiotic or a combination of antibiotics, for example, a combination of zeocin and blasticidin, is/are applied, and antibiotic-resistant colonies are identified and isolated. These are tested for the level of expression of transgenes.
In certain embodiments, provided is a protein expression unit comprising:

    
    
        a bicistronic gene comprising an open reading frame encoding a protein of interest, a protein translation initiation site with a reduced translation efficiency, a selection marker and wherein the bicistronic gene is under control of a functional promoter
        at least one STAR sequence.
    
    


In certain embodiments, the protein expression unit further comprises: a monocistronic gene comprising an open reading frame encoding a second selection marker and wherein the monocistronic gene is under control of a functional promoter.
In certain embodiments, the protein expression unit comprises at least two STAR sequences which are preferentially arranged such that the protein expression unit is flanked on either side by at least one STAR sequence. Examples of such a protein expression unit are provided within the experimental part of this patent application (for example, FIGS. 1 and 5).
In certain embodiments, the protein expression unit described herein comprises STAR sequences, wherein the STAR sequences are essentially identical.
In certain embodiments, provided is a protein expression unit comprising:

    
    
        a bicistronic gene comprising an open reading frame encoding a protein of interest, a protein translation initiation site with a reduced translation efficiency, a selection marker and wherein the bicistronic gene is under control of a functional promoter
        at least one STAR sequence, and is optionally provided with a monocistronic gene cassette, wherein the STAR sequence is depicted in Table 3 and/or SEQ ID NOs:1-119 and/or a functional equivalent and/or a functional fragment thereof and even more preferred wherein the STAR sequence is STAR 18.
    
    


In certain embodiments, a protein expression unit described herein is provided wherein the protein translation initiation site with a reduced translation efficiency comprises an IRES. More preferably, a modified, e.g., weaker, IRES is used.
In certain embodiments, a protein expression unit described herein is provided wherein the protein expression unit is a vector. In certain embodiments, the DNA-carrier is a vector (or plasmid; the terms are used interchangeably herein). In certain embodiments, the vector is a viral vector and in a more preferred embodiment, the viral vector is an adenoviral vector or a retroviral vector. Other viral vectors can also be used in a method described herein.
In certain embodiments, a protein expression unit is provided, wherein the protein of interest is an immunoglobulin heavy chain. In yet another preferred embodiment, a protein expression unit described herein is provided, wherein the protein of interest is an immunoglobulin light chain. When these two protein expression units are present within the same cells or host cell a multimeric protein and more specifically an antibody is assembled.
The invention includes a cell provided with a protein expression unit comprising a STAR.
The invention also includes a cells or host cell comprising at least one protein expression unit described herein. Such a cells or host cell is then, for example, used for large-scale production processes.
Also included is a cell obtainable according to anyone of the methods as described herein. The invention furthermore includes a protein obtainable from the cell (for example, via the process of protein purification). In certain embodiments, the protein is a multimeric protein and even more preferably, the multimeric protein is an antibody. Such an antibody can be used in pharmaceutical and/or diagnostic applications.
The foregoing discussion and the following examples are provided for illustrative purposes, and they are not intended to limit the scope of the invention as claimed herein. They simply provide some of the preferred embodiments of the invention. Modifications and variations, which may occur to one of ordinary skill in the art, are within the intended scope of this invention. Various other embodiments apply to the invention, including: other selectable marker genes; other IRES elements or means of attenuating IRES activity; other elements affecting transcription including promoters, enhancers, introns, terminators, and polyadenylation sites; other orders and/or orientations of the monocistronic and bicistronic genes; other anti-repressor elements or parts, derivations, and/or analogues thereof; other vector systems for delivery of the inventive DNA molecules into eukaryotic host cells; and applications of the inventive method to other transgenic systems.
EXAMPLES
Example 1
STAR Elements and Two-Step Selection Improve the Predictability of Transgene Expression
Improved transgene expression for heterologous protein production is achieved by using a two-step antibiotic selection procedure. The two-step procedure increases the predictability of finding recombinant host cell lines that express the transgene to high levels, thus increasing the yield of the heterologous protein.
Materials and Methods
Plasmid Construction
The pSDH-SIB/Z and pSDH-GIB/Z families of plasmids were constructed as follows: The zeocin selectable marker was recovered by polymerase chain reaction (PCR) amplification from plasmid pEM7/zeo (Invitrogen V500-20) using primers E99 and E100 (SEQ ID NOS:186 and 187, respectively) (all PCR primers and mutagenic oligonucleotide sequences are listed in Table 1), and cloned directionally into the XbaI and NotI sites of multiple cloning site (MCS) B of pIRES (Clontech 6028-1) to create pIRES-zeo. The blasticidin selectable marker was recovered by PCR from plasmid pCMV/bsd (Invitrogen V510-20) using primers E84 and E85 (SEQ ID NOS:176 and 177, respectively), and cloned directionally into the XbaI and NotI sites MCS-B of pIRES to create pIRES-bsd. The SEAP (secreted alkaline phosphatase) reporter gene was recovered by PCR from plasmid pSEAP2-basic (Clontech 6049-1) using primers F11 and E87 (SEQ ID NOS:188 and 178, respectively), and cloned directionally into MCS-A of pIRES-zeo and pIRES-bsd to create plasmids pIRES-SEAP-zeo and pIRES-SEAP-bsd. The GFP reporter gene was recovered from plasmid phr-GFP-1 (Stratagene 240059) by restriction digestion with NheI and EcoRI, and ligated directionally into MCS-A of pIRES-zeo and pIRES-bsd to create plasmids pIRES-GFP-zeo and pIRES-GFP-bsd. A linker was inserted at the non-methylated ClaI site of each of these plasmids (downstream of the neomycin resistance marker) to introduce an AgeI site using oligonucleotides F34 and F35 (SEQ ID NOS:204 and 205, respectively).
The pSDH-Tet vector was constructed by PCR of the luciferase open reading frame from plasmid pREP4-HSF-Luc (van der Vlag et al., 2000) using primers C67 and C68 (SEQ ID NOS:142 and 143, respectively), and insertion of the SacII/BamHI fragment into SacII/BamHI-digested pUHD10-3 (Gossen & Bujard, 1992). The luciferase expression unit was re-amplified with primers C65 and C66 (SEQ ID NOS:140 and 141, respectively), and re-inserted into pUHD10-3 in order to flank it with multiple cloning sites (MCSI and MCSII). An AscI site was then introduced into MCSI by digestion with EcoRI and insertion of a linker (comprised of annealed oligonucleotides D93 and D94, SEQ ID NOS:158 and 159, respectively). The CMV promoter was amplified from plasmid pCMV-Bsd with primers D90 and D91 (SEQ ID NOS:156 and 157, respectively), and used to replace the Tet-Off promoter in pSDH-Tet by SalI/SacII digestion and ligation to create vector pSDH-CMV. The luciferase open reading frame in this vector was replaced by SEAP as follows: vector pSDH-CMV was digested with SacII and BamHI and made blunt; the SEAP open reading frame was isolated from pSEAP-basic by EcoRI/SalI digestion, made blunt and ligated into pSDH-CMV to create vector pSDH-CS. The puromycin resistance gene under control of the SV40 promoter was isolated from plasmid pBabe-Puro (Morgenstern & Land, 1990) by PCR, using primers C81 and C82 (SEQ ID NOS:144 and 145, respectively). This was ligated into vector pGL3-control (BamHI site removed) (Promega E1741) digested with NcoI/XbaI, to create pGL3-puro. pGL3-puro was digested with BglII/SalI to isolate the SV40-puro resistance gene, which was made blunt and ligated into NheI digested, blunt-ended pSDH-CS. The resulting vector, pSDH-CSP, is shown in FIG. 2. STAR18 (SEQ ID NO:18) was inserted into MCSI and MCSII in two steps, by digestion of the STAR element and the pSDH-CSP vector with an appropriate restriction enzyme, followed by ligation. The orientation of the STAR element was determined by restriction mapping. The identity and orientation of the inserts were verified by DNA sequence analysis. Sequencing was performed by the dideoxy method (Sanger et al., 1977) using a Beckman CEQ2000 automated DNA sequencer, according to the manufacturer's instructions. Briefly, DNA was purified from E. coli using QIAprep® Spin Miniprep and Plasmid Midi Kits (QIAGEN® 27106 and 12145, respectively). Cycle sequencing was carried out using custom oligonucleotides C85, E25, and E42 (SEQ ID NOS:146, 173 and 174, respectively) (Table 1), in the presence of dye terminators CEQTM Dye Terminator Cycle Sequencing Kit, Beckman 608000).
pSDH-CSP plasmids containing STAR elements were modified as follows: for receiving SEAP-IRES-zeo/bsd cassettes, an AgeI site was introduced at the BglII site by insertion of a linker, using oligonucleotides F32 and F33 (SEQ ID NOS:202 and 203, respectively); for receiving GFP-IRES-zeo/bsd cassettes, an AgeI site was introduced at the Bsu36I site by insertion of a linker, using oligonucleotides F44 and F45 (SEQ ID NOS:206 and 207, respectively). The SEAP-IRES-zeo/bsd cassettes were inserted into the pSDH-CSP-STAR18 plasmid by replacement of the Bsu36I/AgeI fragment with the corresponding fragments from the pIRES-SEAP-zeo/bsd plasmids. The GFP-IRES-zeo/bsd cassettes were inserted into pSDH-CSP-STAR plasmids by replacement of the BglII/AgeI fragment with the corresponding fragments from the pIRES-GFP-zeo/bsd plasmids. The resulting plasmid families, pSDH-SIB/Z and pSDH-GIB/Z, are shown in FIGS. 3A and 3B, respectively.
All cloning steps were carried out following the instructions provided by the manufacturers of the reagents used, according to methods known in the art (Sambrook et al., 1989).
Transfection and Culture of CHO Cells
The CHO cell line CHO-K1 (ATCC CCL-61) was cultured in HAMS-F12 medium +10% Fetal Calf Serum containing 2 mM glutamine, 100 U/ml penicillin, and 100 micrograms/ml streptomcyin at 37° C., 5% CO2. Cells were transfected with the pSDH-SIZ plasmids using SuperFect® (QIAGEN®) as described by the manufacturer. Briefly, cells were seeded to culture vessels and grown overnight to 70-90% confluence. SuperFect® reagent was combined with plasmid DNA at a ratio of 6 microliters per microgram (e.g., for a 10 cm Petri dish, 20 micrograms DNA and 120 microliters SuperFect®) and added to the cells. After overnight incubation the transfection mixture was replaced with fresh medium, and the transfected cells were incubated further. After overnight cultivation, cells were seeded into fresh culture vessels and 500 micrograms/ml neomycin was added. Neomycin selection was complete within three to four days. Fresh medium was then added containing zeocin (100 μg/ml) and cultured further. Individual clones were isolated after 4-5 days and cultured further. Expression of the reporter gene was assessed by measuring SEAP activity approximately three weeks after transfection.
Secreted Alkaline Phosphatase (SEAP) Assay
SEAP activity (Berger et al., 1988, Henthorn et al., 1988, Kain, 1997, Yang et al., 1997) in the culture media of the clones was determined as described by the manufacturer (Clontech Great EscAPeTM kit #K2041). Briefly, an aliquot of medium was heat inactivated at 65° C., then combined with assay buffer and CSPD chemiluminescent substrate and incubated at room temperature for ten minutes. The rate of substrate conversion was then determined in a luminometer (Turner 20/20TD). Cell density was determined by counting trypsinized cells in a Coulter ACT10 cell counter.
Results
Transfection of the pSDH-SIZ-STAR18 expression vector consistently results in  ̃10-fold more colonies than transfection of the empty pSDH-SIZ vector, presumably due to the increased proportion of primary transfectants that are able to bring the neomycin resistance gene to expression. The outcome of a typical experiment is shown in Table 2, in which transfection of the empty vector yielded  ̃100 G418-resistant colonies, and transfection of the STAR18 vector yielded  ̃1000 colonies.
The expression of the SEAP reporter transgene was compared between the empty pSDH-SIZ vector (hence, without a STAR sequence) and the STAR18 vector FIG. 4). The populations of G418-resistant isolates were divided into two sets. The first set was cultured with G418 only (one-step selection). For this set, the inclusion of STAR18 (SEQ ID NO:18) to protect the transgene from silencing resulted in higher yield of reporter protein: the maximal level of expression among the 20 clones analyzed was 2-3-fold higher than the maximal expression level of clones without the STAR element. The inclusion of STAR18 (SEQ ID NO:18) also led increased predictability: more than 25% of the STAR18 clones had expression levels greater than or equal to the maximum expression level observed in the STARless clones. In this population of STAR18 clones, 70% had expression above the background level, while only 50% of the STARless clones had expression above the background level.
The performance of STAR18 (SEQ ID NO:18) was even better when used in a two-step selection. The second set of G418-resistant isolates was treated with zeocin. Clones that survived the two-step selection regime were assayed for expression of the SEAP reporter transgene. In this case too, the STAR18 (SEQ ID NO:18) element increased the yield compared to the STARless clones by approximately three-fold. The predictability was also increased by inclusion of STAR18 (SEQ ID NO:18):  ̃80% of the population had expression levels greater than the highest-expressing STARless clone.
When the one-step selection is compared with the two-step selection, it can be seen that the latter is superior in terms of both yield and predictability. In fact with two-step selection, no clones appear with background levels of expression. This is due to the requirement imposed on clones that survive zeocin selection that they have high levels of transcription of the bicistronic SEAP-zeocin gene. As indicated in Table 2, the elimination of low-producing clones by the second antibiotic selection step increases the predictability of finding high-producing clones; when STAR18 (SEQ ID NO:18) is included in the expression unit, this increased predictability is improved from three-fold to thirty-fold. In summary, when STAR elements are used in combination with two-step antibiotic selection, the predictability of finding clones with high yields of a transgene is dramatically improved. Application of this increased predictability to two or more transgenes simultaneously will significantly increase the likelihood of finding clones that have high yields of multimeric proteins.
Example 2
Simultaneous Expression of Two Proteins is Improved by Two-Step Selection and STAR Elements
Improved expression of heterologous multimeric proteins, such as antibodies, is achieved. This example demonstrates that the combination of STAR elements and two-step antibiotic selection improves the predictability of establishing recombinant host cell lines that express balanced and proportional amounts of two heterologous polypeptides at high yields. This method of the invention is applicable in practice to multimeric proteins such as antibodies. It is demonstrated in this example using two reporter proteins, secreted alkaline phosphatase (SEAP) and green fluorescent protein (GFP).
Materials and Methods
Plasmids
The pSDH-SIB/Z and pSDH-GIB/Z families of plasmids described in Example 1 are used. Cloning of STAR elements x and y, transfection and culture of host cells, and SEAP assay are described in Example 1. The assay for GFP is performed according to the manufacturer's instructions.
Results
Results show an increased number of clones wherein the two reporter proteins are both expressed. Moreover, expression was balanced in many of such clones.
Example 3
General-Purpose Vectors for Simultaneous Expression of Multiple Polypeptides
The expression system tested and validated in Example 1 has been modified to facilitate its application to any polypeptide that is preferably co-expressed with another polypeptide or polypeptides in a host cell, for example, the heavy and light chains of recombinant antibodies. It is designed for easy and rapid construction of the expression units. This improved system is described in this example.
Materials and Methods
Plasmids
The construction of the plasmids PP1 to PP5 is described below, and their map is shown in FIG. 5. Plasmid pd2EGFP (Clontech 6010-1) was modified by insertion of a linker at the BsiWI site to yield pd2EGFP-link. The linker (made by annealing oligonucleotides F25 and F26, SEQ ID NOS:200 and 201, respectively) introduces sites for the PacI, BglII, and EcoRV restriction endonucleases. This creates the multiple cloning site MCSII for insertion of STAR elements. Then primers F23 and F24 (SEQ ID NOS:198 and 199, respectively) were used to amplify a region of 0.37 kb from pd2EGFP, which was inserted into the BglII site of pIRES (Clontech 6028-1) to yield pIRES-stuf. This introduces sites for the AscI and SwaI restriction endonucleases at MCSI, and acts as a “stuffer fragment” to avoid potential interference between STAR elements and adjacent promoters. pIRES-stuf was digested with BglII and FspI to liberate a DNA fragment composed of the stuffer fragment, the CMV promoter, the IRES element (flanked by multiple cloning sites MCS A and MCS B), and the SV40 polyadenylation signal. This fragment was ligated with the vector backbone of pd2EGFP-link produced by digestion with BamHI and StuI, to yield pd2IRES-link.
The open reading frames of the zeocin-, neomycin, or puromycin-resistance genes were inserted into the BamHI/NotI sites of MCS B in pd2IRES-link as follows: the zeocin-resistance ORF was amplified by PCR with primers F18 and E100 (SEQ ID NOS:193 and 187, respectively) from plasmid pEM7/zeo, digested with BamHI and NotI, and ligated with BamHI/NotI-digested pd2IRES-link to yield pd2IRES-link-zeo. The neomycin-resistance ORF was amplified by PCR with primers F19 and F20 (SEQ ID NOS:194 and 195, respectively) from pIRES, digested with BamHI and NotI, and ligated with BamHI/NotI-digested pd2IRES-link to yield pd2IRES-link-neo. The puromycin-resistance ORF was amplified by PCR with primers F21 and F22 (SEQ ID NOS:196 and 197, respectively) from plasmid pBabe-Puro (Morgenstern & Land, 1990), digested with BamHI and NotI, and ligated with BamHI/NotI-digested pd2IRES-link to yield pd2IRES-link-puro.
The GFP reporter ORF was introduced into pd2IRES-link-puro by amplification of phr-GFP-1 with primers F16 and F17 (SEQ ID NOS:191 and 192, respectively), and insertion of the EcoRI-digested GFP cassette into the EcoRI site in MCS A of the pd2IRES-link-puro plasmid, to yield plasmid PP1 (FIG. 5A). Correct orientation was verified by restriction mapping. The SEAP reporter ORF was introduced into pd2IRES-link-zeo and pd2IRES-link-neo by PCR amplification of pSEAP2-basic with primers F14 and F15 (SEQ ID NOS:189 and 190, respectively), and insertion of the EcoRI-digested SEAP cassette into the EcoRI sites in MCS A of the plasmids pd2IRES-link-zeo (to yield plasmid PP2, FIG. 5B) and pd2IRES-link-neo (to yield plasmid PP3, FIG. 5C). Correct orientation was verified by restriction mapping.
Plasmids PP1, PP2 and PP3 contain a bicistronic gene for expression of a reporter protein and an antibiotic resistance marker. In order to carry out two-step antibiotic selection with separate antibiotics, a monocistronic resistance marker was introduced as follows: pIRES-stuf was digested with ClaI, made blunt with Klenow enzyme, and digested further with BglII. This liberated a DNA fragment composed of the stuffer fragment, the CMV promoter, the IRES element (flanked by multiple cloning sites MCS A and MCS B), the SV40 polyadenylation signal, and the neomycin resistance marker under control of the SV40 promoter. This fragment was ligated with the vector backbone of pd2EGFP-link produced by digestion with BamHI and StuI, to yield pd2IRES-link-neo. Then as described above the GFP and puro cassettes were introduced to yield PP4 (FIG. 5D), and the SEAP and zeo cassettes were introduced to yield PP5 (FIG. 5E).
Example 4
Predictability and Yield are Improved by Application of STAR Elements in Expression Systems
STAR elements function to block the effect of transcriptional repression influences on transgene expression units. These repression influences can be due to heterochromatin (“position effects” (Boivin & Dura, 1998)) or to adjacent copies of the transgene (“repeat-induced gene silencing” (Garrick et al., 1998)). Two of the benefits of STAR elements for protein production are increased predictability of finding high-expressing primary recombinant host cells, and increased yield during production cycles. These benefits are illustrated in this example.
Materials and Methods
Construction of the pSDH vectors and STAR-containing derivatives: The pSDH-Tet vector was constructed by polymerase chain reaction amplification (PCR). of the luciferase open reading frame from plasmid pREP4-HSF-Luc (van der Vlag et al., 2000) using primers C67 and C68 (SEQ ID NOS:142 and 143, respectively) (all PCR primers and mutagenic oligonucleotides are listed in Table 1), and insertion of the SacII/BamHI fragment into SacII/BamHI-digested pUHD10-3 (Gossen & Bujard, 1992). The luciferase expression unit was re-amplified with primers C65 and C66 (SEQ ID NOS:140 and 141, respectively), and re-inserted into pUHD10-3 in order to flank it with two multiple cloning sites (MCSI and MCSII). An AscI site was then introduced into MCSI by digestion with EcoRI and insertion of a linker (comprised of annealed oligonucleotides D93 and D94, SEQ ID NOS:158 and 159, respectively). The CMV promoter was amplified from plasmid pCMV-Bsd (Invitrogen K510-01) with primers D90 and D91 (SEQ ID NOS:156 and 157, respectively), and used to replace the Tet-Off promoter in pSDH-Tet by SalI/SacII digestion and ligation to create vector pSDH-CMV. The luciferase open reading frame in this vector was replaced by SEAP (Secreted Alkaline Phosphatase) as follows: vector pSDH-CMV was digested with SacII and BamHI and made blunt; the SEAP open reading frame was isolated from pSEAP-basic (Clontech 6037-1) by EcoRI/SalI digestion, made blunt and ligated into pSDH-CMV to create vector pSDH-CS. The puromycin resistance gene under control of the SV40 promoter was isolated from plasmid pBabe-Puro (Morgenstern & Land, 1990) by PCR, using primers C81 and C82 (SEQ ID NOS:144 and 145, respectively). This was ligated into vector pGL3-control (BamHI site removed) (Promega E1741) digested with NcoI/XbaI, to create pGL3-puro. pGL3-puro was digested with BglII/SalI to isolate the SV40-puro resistance gene, which was made blunt and ligated into NheI digested, blunt-ended pSDH-CS. The resulting vector, pSDH-CSP, is shown in FIG. 6. All cloning steps were carried out following the instructions provided by the manufacturers of the reagents, according to methods known in the art (Sambrook et al., 1989).
STAR elements were inserted into MCSI and MCSII in two steps, by digestion of the STAR element and the pSDH-CSP vector with an appropriate restriction enzyme, followed by ligation. The orientation of STAR elements in recombinant pSDH vectors was determined by restriction mapping. The identity and orientation of the inserts were verified by DNA sequence analysis. Sequencing was performed by the dideoxy method (Sanger et al., 1977) using a Beckman CEQTM 2000 automated DNA sequencer, according to the manufacturer's instructions. Briefly, DNA was purified from E. coli using QIAprep® Spin Miniprep and Plasmid Midi Kits (QIAGEN® 27106 and 12145, respectively). Cycle sequencing was carried out using custom oligonucleotides C85, E25, and E42 (SEQ ID NOS:146, 173 and 174, respectively) (Table 1), in the presence of dye terminators (CEQTM Dye Terminator Cycle Sequencing Kit, Beckman 608000).
Transfection and Culture of CHO Cells with pSDH Plasmids
The Chinese Hamster Ovary cell line CHO-K1 (ATCC CCL-61) was cultured in HAMS-F12 medium +10% Fetal Calf Serum containing 2 mM glutamine, 100 U/ml penicillin, and 100 micrograms/ml streptomcyin at 37° C., 5% CO2. Cells were transfected with the pSDH-CSP vector, and its derivatives containing STAR6 (SEQ ID NO:6) or STAR49 (SEQ ID NO:49) in MCSI and MCSII, using SuperFect® (QIAGEN®) as described by the manufacturer. Briefly, cells were seeded to culture vessels and grown overnight to 70-90% confluence. SuperFect® reagent was combined with plasmid DNA (linearized in this example by digestion with PvuI) at a ratio of 6 microliters per microgram (e.g., for a 10 cm Petri dish, 20 micrograms DNA and 120 microliters SuperFect®) and added to the cells. After overnight incubation the transfection mixture was replaced with fresh medium, and the transfected cells were incubated further. After overnight cultivation, 5 micrograms/ml puromycin was added. Puromycin selection was complete in two weeks, after which time individual puromycin resistant CHO/pSDH-CSP clones were isolated at random and cultured further.
Secreted Alkaline Phosphatase (SEAP) Assay
SEAP activity (Berger et al., 1988, Henthorn et al., 1988, Kain, 1997, Yang et al., 1997) in the culture medium of CHO/pSDH-CSP clones was determined as described by the manufacturer (Clontech Great EscAPeTM kit #K2041). Briefly, an aliquot of medium was heat inactivated at 65° C., then combined with assay buffer and CSPD chemiluminescent substrate and incubated at room temperature for ten minutes. The rate of substrate conversion was then determined in a luminometer (Turner 20/20TD). Cell density was determined by counting trypsinized cells in a Coulter ACT10 cell counter.
Transfection and Culture of U-2 OS Cells with pSDH Plasmids
The human osteosarcoma U-2 OS cell line (ATCC #HTB-96) was cultured in Dulbecco's Modified Eagle Medium +10% Fetal Calf Serum containing glutamine, penicillin, and streptomycin (supra) at 37° C., 5% CO2. Cells were co-transfected with the pSDH-CMV vector, and its derivatives containing STAR6 (SEQ ID NO:6) or STAR8 (SEQ ID NO:8) in MCSI and MCSII, (along with plasmid pBabe-Puro) using SuperFect® (supra). Puromycin selection was complete in two weeks, after which time individual puromycin resistant U-2 OS/pSDH-CMV clones were isolated at random and cultured further.
Luciferase Assay
Luciferase activity (Himes & Shannon, 2000) was assayed in resuspended cells according to the instructions of the assay kit manufacturer (Roche 1669893), using a luminometer (Turner 20/20TD). Total cellular protein concentration was determined by the bicinchoninic acid method according to the manufacturer's instructions (Sigma B-9643), and used to normalize the luciferase data.
Results
Recombinant CHO cell clones containing the pSDH-CSP vector, or pSDH-CSP plasmids containing STAR6 (SEQ ID NO:6) or STAR49 (SEQ ID NO:49) (Table 6), were cultured for three weeks. The SEAP activity in the culture supernatants was then determined, and is expressed on the basis of cell number (FIG. 7). As can be seen, clones with STAR elements in the expression units were isolated that express 2-3 fold higher SEAP activity than clones whose expression units do not include STAR elements. Furthermore, the number of STAR-containing clones that express SEAP activity at or above the maximal activity of the STAR-less clones is quite high: 25% to 40% of the STAR clone populations exceed the highest SEAP expression of the pSDH-CSP clones.
Recombinant U-2 OS cell clones containing the pSDH-CMV vector, or pSDH-CMV plasmids containing STAR6 (SEQ ID NO:6) or STAR8 (SEQ ID NO:8) (Table 6), were cultured for three weeks. The luciferase activity in the host cells was then determined, and is expressed as relative luciferase units (FIG. 8), normalized to total cell protein. The recombinant U-2 OS clones with STAR elements flanking the expression units had higher yields than the STAR-less clones: the highest expression observed from STAR8 clones was 2-3 fold higher than the expression from STAR-less clones. STAR6 clones had maximal expression levels five-fold higher than the STAR-less clones. The STAR elements conferred greater predictability as well: for both STAR elements, 15 to 20% of the clones displayed luciferase expression at levels comparable to or greater than the STAR-less clone with the highest expression level.
These results demonstrate that, when used with the strong CMV promoter, STAR elements increase the yield of heterologous proteins (luciferase and SEAP). All three of the STAR elements introduced in this example provide elevated yields. The increased predictability conferred by the STAR elements is manifested by the large proportion of the clones with yields equal to or greater than the highest yields displayed by the STAR-less clones.
Example 5
STAR Elements Improve the Stability of Transgene Expression
During cultivation of recombinant host cells, it is common practice to maintain antibiotic selection. This is intended to prevent transcriptional silencing of the transgene, or loss of the transgene from the genome by processes such as recombination. However it is undesirable for production of proteins, for a number of reasons. First, the antibiotics that are used are quite expensive, and contribute significantly to the unit cost of the product. Second, for biopharmaceutical use, the protein must be demonstrably pure, with no traces of the antibiotic in the product. One advantage of STAR elements for heterologous protein production is that they confer stable expression on transgenes during prolonged cultivation, even in the absence of antibiotic selection; this property is demonstrated in this example.
Materials and Methods
The U-2 OS cell line was transfected with the plasmid pSDH-Tet-STAR6 and cultivated as described in Example 4. Individual puromycin-resistant clones were isolated and cultivated further in the absence of doxycycline. At weekly intervals the cells were transferred to fresh culture vessels at a dilution of 1:20. Luciferase activity was measured at periodic intervals as described in Example 4. After 15 weeks the cultures were divided into two replicates; one replicate continued to receive puromycin, while the other replicate received no antibiotic for the remainder of the experiment (25 weeks total).
Results
Table 7 presents the data on luciferase expression by an expression unit flanked with STAR6 (SEQ ID NO:6) during prolonged growth with or without antibiotic. As can be seen, the expression of the reporter transgene, luciferase, remains stable in the U-2 OS host cells for the duration of the experiment. After the cultures were divided into two treatments (plus antibiotic and without antibiotic) the expression of luciferase was essentially stable in the absence of antibiotic selection. This demonstrates the ability of STAR elements to protect transgenes from silencing or loss during prolonged cultivation. It also demonstrates that this property is independent of antibiotic selection. Therefore, production of proteins is possible without incurring the costs of the antibiotic or of difficult downstream processing.
Example 6
Minimal Essential Sequences of STAR Elements
STAR elements are isolated from the genetic screen as described herein. The screen uses libraries constructed with human genomic DNA that was size-fractionated to approximately 0.5-2 kilobases (supra). The STAR elements range from 500 to 2361 base pairs (Table 6). It is likely that, for many of the STAR elements that have been isolated, STAR activity is conferred by a smaller DNA fragment than the initially isolated clone. It is useful to determine these minimum fragment sizes that are essential for STAR activity, for two reasons. First, smaller functional STAR elements would be advantageous in the design of compact expression vectors, since smaller vectors transfect host cells with higher efficiency. Second, determining minimum essential STAR sequences permits the modification of those sequences for enhanced functionality. Two STAR elements have been fine-mapped to determine their minimal essential sequences.
Materials and Methods:
STAR10 (SEQ ID NO:10) (1167 base pairs) and STAR27 (SEQ ID NO:27) (1520 base pairs) have been fine-mapped. They have been amplified by PCR to yield sub-fragments of approximately equal length (FIG. 9 legend). For initial testing, these have been cloned into the pSelect vector at the BamHI site, and transfected into U-2 OS/Tet-Off/LexA-HP1 cells. The construction of the host strains has been described (van der Vlag et al., 2000). Briefly, they are based on the U-2 OS human osteosarcoma cell line (American Type Culture Collection HTB-96). U-2 OS is stably transfected with the pTet-Off plasmid (Clontech K1620-A), encoding a protein chimera consisting of the Tet-repressor DNA binding domain and the VP16 transactivation domain. The cell line is subsequently stably transfected with fusion protein genes containing the LexA DNA binding domain, and the coding regions of either HP1 or HPC2 (two Drosophila Polycomb group proteins that repress gene expression when tethered to DNA). The LexA-repressor genes are under control of the Tet-Off transcriptional regulatory system (Gossen and Bujard, 1992). After selection for hygromycin resistance, LexA-HP1 was induced by lowering the doxycycline concentration. Transfected cells were then incubated with zeocin to test the ability of the STAR fragments to protect the SV40-Zeo expression unit from repression due to LexA-HP1 binding.
Results
In this experiment STAR10 (SEQ ID NO:10) and STAR27 (SEQ ID NO:27) confer good protection against gene silencing, as expected (FIG. 9). This is manifested by robust growth in the presence of zeocin.
Of the three STAR10 (SEQ ID NO:10) sub-fragments, 10A (corresponding in sequence to roughly the first 400 nucleotides of SEQ ID NO:10) ( ̃400 base pairs) confers on transfected cells vigorous growth in the presence of zeocin, exceeding that of the full-length STAR element. Cells transfected with pSelect constructs containing the other two sub-fragments do not grow in the presence of zeocin. These results identify the  ̃400 base pair 10A fragment as encompassing the DNA sequence responsible for the anti-repression activity of STAR10 (SEQ ID NO:10).
STAR27 (SEQ ID NO:27) confers moderate growth in zeocin to transfected cells in this experiment (FIG. 9). One of the sub-fragments of this STAR, 27B (corresponding in sequence to roughly the second 500 nucleotides of SEQ ID NO:27) ( ̃500 base pairs), permits weak growth of the host cells in zeocin-containing medium. This suggests that the anti-repression activity of this STAR is partially localized on sub-fragment 27B (corresponding in sequence to roughly the first 500 nucleotides of SEQ ID NO:27), but full activity requires sequences from 27A (corresponding in sequence to roughly the first 500 nucleotides of SEQ ID NO:27) and/or 27C (corresponding in sequence to roughly the third 500 nucleotides of SEQ ID NO:27) (each 500 base pairs) as well.
Example 7
STAR Elements Function in Diverse Strains of Cultured Mammalian Cells
The choice of host cell line for (heterologous) protein expression is a critical parameter for the quality, yield, and unit cost of the protein. Considerations such as post-translational modifications, secretory pathway capacity, and cell line immortality dictate the appropriate cell line for a particular biopharmaceutical production system. For this reason, the advantages provided by STAR elements in terms of yield, predictability, and stability should be obtainable in diverse cell lines. This was tested by comparing the function of STAR6 (SEQ ID NO:6) in the human U-2 OS cell line in which it was originally cloned, and the CHO cell line which is widely applied in biotechnology.
Materials and Methods:
See, the experiments of Example 4.
Results
The expression of the SEAP reporter gene in CHO cells is presented in FIG. 7; the expression of the luciferase reporter gene in U-2 OS cells is presented in FIG. 8. By comparison of the results of these two experiments, it is apparent that the STAR6 (SEQ ID NO:6) element is functional in both cell lines: reporter gene expression was more predictable in both of them, and clones of each cell line displayed higher yields, when the reporter gene was shielded from position effects by STAR6 (SEQ ID NO:6). These two cell lines are derived from different species (human and hamster) and different tissue types (bone and ovary), reflecting the broad range of host cells in which this STAR element can be utilized in improving heterologous protein expression.
Example 8
STAR Elements Function in the Context of Various Transcriptional Promoters
Transgene transcription is achieved by placing the transgene open reading frame under control of an exogenous promoter. The choice of promoter is influenced by the nature of the (heterologous) protein and the production system. In most cases, strong constitutive promoters are preferred because of the high yields they can provide. Some viral promoters have these properties; the promoter/enhancer of the cytomegalovirus immediate early gene (“CMV promoter”) is generally regarded as the strongest promoter in common biotechnological use (Boshart et al., 1985, Doll et al., 1996, Foecking & Hofstetter, 1986). The simian virus SV40 promoter is also moderately strong (Boshart et al., 1985, Foecking & Hofstetter, 1986) and is frequently used for ectopic expression in mammalian cell vectors. The Tet-Off promoter is inducible: the promoter is repressed in the presence of tetracycline or related antibiotics (doxycycline is commonly used) in cell-lines which express the tTA plasmid (Clontech K1620-A), and removal of the antibiotic results in transcriptional induction (Deuschle et al., 1995, Gossen & Bujard, 1992, Izumi & Gilbert, 1999, Umana et al., 1999).
Materials and Methods:
The construction of the pSDH-Tet and pSDH-CMV vectors is described in Example 4. pSDH-SV40 is, amongst others, derived from pSelect-SV40-zeo. The selection vector for STAR elements, pSelect-SV40-zeo is constructed as follows: the pREP4 vector (Invitrogen V004-50) is used as the plasmid backbone. It provides the Epstein Barr oriP origin of replication and EBNA-1 nuclear antigen for high-copy episomal replication in primate cell lines; the hygromycin resistance gene with the thymidine kinase promoter and polyadenylation site, for selection in mammalian cells; and the ampicillin resistance gene and colEl origin of replication for maintenance in Escherichia coli. The vector contains four consecutive LexA operator sites between XbaI and NheI restriction sites (Bunker and Kingston, 1994). Embedded between the LexA operators and the NheI site is a polylinker consisting of the following restriction sites: HindIII-AscI-BamHI-AscI-HindIII. Between the NheI site and a SalI site is the zeocin resistance gene with the SV40 promoter and polyadenylation site, derived from pSV40/Zeo (Invitrogen V502-20); this is the selectable marker for the STAR screen. pSDH-SV40 was constructed by PCR amplification of the SV40 promoter (primers D41 and D42) from plasmid pSelect-SV40-Zeo, followed by digestion of the PCR product with SacII and SalI. The pSDH-CMV vector was digested with SacII and SalI to remove the CMV promoter, and the vector and SV40 fragment were ligated together to create pSDH-SV40. STAR6 (SEQ ID NO:6) was cloned into MCSI and MCSII as described in Example 4. The plasmids pSDH-Tet, pSDH-Tet-STAR6, pSDH-Tet-STAR7, pSDH-SV40 and pSDH-SV40-STAR6 were co-transfected with pBabe-Puro into U-2 OS using SuperFect as described by the manufacturer. Cell cultivation, puromycin selection, and luciferase assays were carried out as described in Example 4.
Results
FIGS. 9, 11, and 12 compare the expression of the luciferase reporter gene from three different promoters: two strong and constitutive viral promoters (CMV and SV40), and the inducible Tet-Off promoter. All three promoters were tested in the context of the STAR6 (SEQ ID NO:6) element in U-2 OS cells. The results demonstrate that the yield and predictability from all three promoters are increased by STAR6 (SEQ ID NO:6). As described in Examples 4 and 7, STAR6 (SEQ ID NO:6) is beneficial in the context of the CMV promoter (FIG. 8). Similar improvements are seen in the context of the SV40 promoter (FIG. 10): the yield from the highest-expressing STAR6 clone is 2-3 fold greater than the best pSDH-SV40 clones, and six STAR clones (20% of the population) have yields higher than the best STAR-less clones. In the context of the Tet-Off promoter under inducing (low doxycycline) concentrations, STAR6 (SEQ ID NO:6) also improves the yield and predictability of transgene expression (FIG. 11): the highest-expressing STAR6 clone has a 20-fold higher yield than the best pSDH-Tet clone, and nine STAR6 clones (35% of the population) have yields higher than the best STAR-less clone. It is concluded that this STAR element is versatile in its transgene-protecting properties, since it functions in the context of various biotechnologically useful promoters of transcription.
Example 9
STAR Element Function can be Directional
While short nucleic acid sequences can be symmetrical (e.g., palindromic), longer naturally-occurring sequences are typically asymmetrical. As a result, the information content of nucleic acid sequences is directional, and the sequences themselves can be described with respect to their 5′ and 3′ ends. The directionality of nucleic acid sequence information affects the arrangement in which recombinant DNA molecules are assembled using standard cloning techniques known in the art (Sambrook et al., 1989). STAR elements are long, asymmetrical DNA sequences, and have a directionality based on the orientation in which they were originally cloned in the pSelect vector. In the examples given above, using two STAR elements in pSDH vectors, this directionality was preserved. This orientation is described as the native or 5′-3′ orientation, relative to the zeocin resistance gene (see FIG. 12). In this example, the importance of directionality for STAR function is tested in the pSDH-Tet vector. Since the reporter genes in the pSDH vectors are flanked on both sides by copies of the STAR element of interest, the orientation of each STAR copy must be considered. This example compares the native orientation with the opposite orientation (FIG. 12).
Materials and Methods:
The STAR66 (SEQ ID NO:66) element was cloned into pSDH-Tet as described in Example 4. U-2 OS cells were co-transfected with plasmids pSDH-Tet-STAR66-native and pSDH-Tet-STAR66-opposite, and cultivated as described in Example 4. Individual clones were isolated and cultivated; the level of luciferase expression was determined as described (supra).
Results
The results of the comparison of STAR66 (SEQ ID NO:66) activity in the native orientation and the opposite orientation are shown in FIG. 13. When STAR66 (SEQ ID NO:66) is in the opposite orientation, the yield of only one clone is reasonably high (60 luciferase units). In contrast, the yield of the highest-expressing clone when STAR66 (SEQ ID NO:66) is in the native orientation is considerably higher (100 luciferase units), and the predictability is much higher as well: seven clones of the native-orientation population (30%) express luciferase above the level of the highest-expressing clone from the opposite-orientation population, and 15 of the clones in the native-orientation population (60%) express luciferase above ten relative luciferase units.
Therefore, it is demonstrated that STAR66 (SEQ ID NO:66) function is directional.
Example 10
Transgene Expression in the Context of STAR Elements is Copy Number-Dependent
Transgene expression units for (heterologous) protein expression are generally integrated into the genome of the host cell to ensure stable retention during cell division. Integration can result in one or multiple copies of the expression unit being inserted into the genome; multiple copies may or may not be present as tandem arrays. The increased yield demonstrated for transgenes protected by STAR elements (supra) suggests that STAR elements are able to permit the transgene expression units to function independently of influences on transcription associated with the site of integration in the genome (independence from position effects (Boivin & Dura, 1998). It suggests further that the STAR elements permit each expression unit to function independently of neighboring copies of the expression unit when they are integrated as a tandem array (independence from repeat-induced gene silencing (Garrick et al., 1998)). Copy number-dependence is determined from the relationship between transgene expression levels and copy number, as described in the example below.
Materials and Methods:
U-2 OS cells were co-transfected with pSDH-Tet-STAR10 and cultivated under puromycin selection as described (supra). Eight individual clones were isolated and cultivated further. Then cells were harvested, and one portion was assayed for luciferase activity as described (supra). The remaining cells were lysed and the genomic DNA purified using the DNeasy® Tissue Kit (QIAGEN® 69504) as described by the manufacturer. DNA samples were quantitated by UV spectrophotometry. Three micrograms of each genomic DNA sample were digested with PvuII and XhoI overnight as described by the manufacturer (New England Biolabs), and resolved by agarose gel electrophoresis. DNA fragments were transferred to a nylon membrane as described (Sambrook et al., 1989), and hybridized with a radioactively labeled probe to the luciferase gene (isolated from BamHI/SacII-digested pSDH-Tet). The blot was washed as described (Sambrook et al., 1989) and exposed to a phosphorimager screen (Personal F/X, BioRad). The resulting autoradiogram (FIG. 14) was analyzed by densitometry to determine the relative strength of the luciferase DNA bands, which represents the transgene copy number.
Results
The enzyme activities and copy numbers (DNA band intensities) of luciferase in the clones from the pSDH-Tet-STAR10 clone population is shown in FIG. 15. The transgene copy number is highly correlated with the level of luciferase expression in these pSDH-Tet-STAR10 clones (r=0.86). This suggests that STAR10 (SEQ ID NO:10) confers copy number-dependence on the transgene expression units, making transgene expression independent of other transgene copies in tandem arrays, and independent of gene-silencing influences at the site of integration.
Example 11
STAR Elements Function as Enhancer Blockers But not Enhancers
Gene promoters are subject to both positive and negative influences on their ability to initiate transcription. An important class of elements that exert positive influences are enhancers. Enhancers are characteristically able to affect promoters even when they are located far away (many kilobase pairs) from the promoter. Negative influences that act by heterochromatin formation (e.g., Polycomb group proteins) have been described above, and these are the target of STAR activity. The biochemical basis for enhancer function and for heterochromatin formation is fundamentally similar, since they both involve binding of proteins to DNA. Therefore, it is important to determine whether STAR elements are able to block positive influences as well as negative influences, in other words, to shield transgenes from genomic enhancers in the vicinity of the site of integration. The ability to shield transgenes from enhancer activity ensures stable and predictable performance of transgenes in biotechnological applications. This example examines the performance of STAR elements in an enhancer-blocking assay.
Another feature of STAR activity that is important to their function is the increased yield they confer on transgenes (Example 4). STARs are isolated on the basis of their ability to maintain high levels of zeocin expression when heterochromatin-forming proteins are bound adjacent to the candidate STAR elements. High expression is predicted to occur because STARs are anticipated to block the spread of heterochromatin into the zeocin expression unit. However, a second scenario is that the DNA fragments in zeocin-resistant clones contain enhancers. Enhancers have been demonstrated to have the ability to overcome the repressive effects of Polycomb-group proteins such as those used in the method of the STAR screen (Zink & Paro, 1995). Enhancers isolated by this phenomenon would be considered false positives, since enhancers do not have the properties claimed here for STARs. In order to demonstrate that STAR elements are not enhancers, they have been tested in an enhancer assay.
The enhancer-blocking assay and the enhancer assay are methodologically and conceptually similar. The assays are shown schematically in FIG. 16. The ability of STAR elements to block enhancers is performed using the E47/E-box enhancer system. The E47 protein is able to activate transcription by promoters when it is bound to an E-box DNA sequence located in the vicinity of those promoters (Quong et al., 2002). E47 is normally involved in regulation of B and T lymphocyte differentiation (Quong et al., 2002), but it is able to function in diverse cell types when expressed ectopically (Petersson et al., 2002). The E-box is a palindromic DNA sequence, CANNTG (SEQ ID NO:120) (Knofler et al., 2002). In the enhancer-blocking assay, an E-box is placed upstream of a luciferase reporter gene (including a minimal promoter) in an expression vector. A cloning site for STAR elements is placed between the E-box and the promoter. The E47 protein is encoded on a second plasmid. The assay is performed by transfecting both the E47 plasmid and the luciferase expression vector into cells; the E47 protein is expressed and binds to the E-box, and the E47/E-box complex is able to act as an enhancer. When the luciferase expression vector does not contain a STAR element, the E47/E-box complex enhances luciferase expression (FIG. 16A, situation 1). When STAR elements are inserted between the E-box and the promoter, their ability to block the enhancer is demonstrated by reduced expression of luciferase activity (FIG. 16A, situation 2); if STARs cannot block enhancers, luciferase expression is activated (FIG. 16A, situation 3).
The ability of STAR elements to act as enhancers utilizes the same luciferase expression vector. In the absence of E47, the E-box itself does not affect transcription. Instead, enhancer behavior by STAR elements will result in activation of luciferase transcription. The assay is performed by transfecting the luciferase expression vector without the E47 plasmid. When the expression vector does not contain STAR elements, luciferase expression is low (FIG. 16B, situation 1). If STAR elements do not have enhancer properties, luciferase expression is low when a STAR element is present in the vector (FIG. 16B, situation 2). If STAR elements do have enhancer properties, luciferase expression will be activated in the STAR-containing vectors (FIG. 16B, situation 3).
Materials and Methods:
The luciferase expression vector was constructed by inserting the E-box and a human alkaline phosphatase minimal promoter from plasmid mu-E5+E2×6-cat(x) (Ruezinsky et al., 1991) upstream of the luciferase gene in plasmid pGL3-basic (Promega E1751), to create pGL3-E-box-luciferase (gift of W. Romanow). The E47 expression plasmid contains the E47 open reading frame under control of a beta-actin promoter in the pHBAPr-1-neo plasmid; E47 in constitutively expressed from this plasmid (gift of W. Romanow).
STAR elements 1, 2, 3, 6, 10, 11, 18, and 27 (SEQ ID NOS:1, 2, 3, 6, 10, 11, 18 and 27, respectively) have been cloned into the luciferase expression vector. Clones containing the Drosophila scs element and the chicken beta-globin HS4-6× core (“HS4”) element have been included as positive controls (they are known to block enhancers, and to have no intrinsic enhancer properties (Chung et al., 1993, Kellum & Schedl, 1992)), and the empty luciferase expression vector has been included as a negative control. All assays were performed using the U-2 OS cell line. In the enhancer-blocking assay, the E47 plasmid was co-transfected with the luciferase expression vectors (empty vector, or containing STAR or positive-control elements). In the enhancer assay, the E47 plasmid was co-transfected with STARless luciferase expression vector as a positive control for enhancer activity; all other samples received a mock plasmid during co-transfection. The transiently transfected cells were assayed for luciferase activity 48 hours after plasmid transfection (supra). The luciferase activity expressed from a plasmid containing no E-box or STAR/control elements was subtracted, and the luciferase activities were normalized to protein content as described (supra).
Results
FIG. 17 shows the results of the enhancer-blocking assay. In the absence of STAR elements (or the known enhancer-blocking elements scs and HS4), the E47/E-box enhancer complex activates expression of luciferase (“vector”); this enhanced level of expression has been normalized to 100. Enhancer activity is blocked by all STAR elements tested. Enhancer activity is also blocked by the HS4 and scs elements, as expected (Bell et al., 2001, Gerasimova & Corces, 2001). These results demonstrate that in addition to their ability to block the spreading of transcriptional silencing (negative influences), STAR elements are able to block the action of enhancers (positive influences).
FIG. 18 shows the results of the enhancer assay. The level of luciferase expression due to enhancement by the E47/E-box complex is set at 100 (“E47”). By comparison, none of the STAR elements bring about significant activation of luciferase expression. As expected, the scs and HS4 elements also do not bring about activation of the reporter gene. Therefore, it is concluded that at least the tested STAR elements do not possess enhancer properties.
Example 12
STAR Elements are Conserved Between Mouse and Human
BLAT analysis of the STAR DNA sequence against the human genome database (WorldWideWeb.genome.ucsc.edu/cgi-bin/hgGateway) reveals that some of these sequences have high sequence conservation with other regions of the human genome. These duplicated regions are candidate STAR elements; if they do show STAR activity, they would be considered paralogs of the cloned STARs (two genes or genetic elements are said to be paralogous if they are derived from a duplication event (Li, 1997)).
BLAST analysis of the human STARs against the mouse genome (WorldWideWeb.ensembl.org/Mus_musculus/blastview) also reveals regions of high sequence conservation between mouse and human. This sequence conservation has been shown for fragments of 15 out of the 65 human STAR elements. The conservation ranges from 64% to 89%, over lengths of 141 base pairs to 909 base pairs (Table 8). These degrees of sequence conservation are remarkable and suggest that these DNA sequences may confer STAR activity within the mouse genome as well. Some of the sequences from the mouse and human genomes in Table 8 could be strictly defined as orthologs (two genes or genetic elements are said to be orthologous if they are derived from a speciation event (Li, 1997)). For example, STAR6 (SEQ ID NO:6) is between the SLC8 A1 and HAAO genes in both the human and mouse genomes. In other cases, a cloned human STAR has a paralog within the human genome, and its ortholog has been identified in the mouse genome. For example, STAR3a (SEQ ID NO:3) is a fragment of the 15q11.2 region of human chromosome 15. This region is 96.9% identical (paralogous) with a DNA fragment at 5q33.3 on human chromosome 5, which is near the IL12B interleukin gene. These human DNAs share approximately 80% identity with a fragment of the 11B2 region on mouse chromosome 11. The 11B2 fragment is also near the (mouse) IL12B interleukin gene. Therefore, STAR3a (SEQ ID NO:3) and the mouse 11B2 fragment can be strictly defined as paralogs.
In order to test the hypothesis that STAR activity is shared between regions of high sequence conservation in the mouse and human genome, one of the human STARs with a conserved sequence in mouse, STAR18 (SEQ ID NO:18), has been analyzed in greater detail. The sequence conservation in the mouse genome detected with the original STAR18 clone extends leftward on human chromosome 2 for about 500 base pairs (FIG. 19; left and right relate to the standard description of the arms of chromosome 2). In this example, we examine whether the region of sequence conservation defines a “naturally occurring” STAR element in human that is more extensive in length than the original clone. We also examine whether the STAR function of this STAR element is conserved between mouse and human.
Materials and Methods
The region of mouse/human sequence conservation around STAR 18 (SEQ ID NO:18) was recovered from human BAC clone RP11-387A1 by PCR amplification, in three fragments: the entire region (primers E93 and E94, SEQ ID NOS:180 and 181, respectively), the leftward half (primers E93 and E92, SEQ ID NOS:180 and 179, respectively), and the rightward half (primers E57 and E94, SEQ ID NOS:175 and 181, respectively). The corresponding fragments from the homologous mouse region were recovered from BAC clone RP23-400H17 in the same fashion (primers E95 (SEQ ID NO:182) and E98 (SEQ ID NO:185), E95 (SEQ ID NO:182) and E96 (SEQ ID NO:183), and E97 (SEQ ID NO:184) and E98 (SEQ ID NO:185), respectively). All fragments were cloned into the pSelect vector and transfected into a U-2 OS/Tet-Off/LexA-HP1 cell line (supra). Following transfection, hygromycin selection was carried out to select for transfected cells. The LexA-HP1 protein was induced by lowering the doxycycline concentration, and the ability of the transfected cells to withstand the antibiotic zeocin (a measure of STAR activity) was assessed by monitoring cell growth.
Results
The original STAR18 clone was isolated from Sau3AI digested human DNA ligated into the pSelect vector on the basis of its ability to prevent silencing of a zeocin resistance gene. Alignment of the human STAR18 clone (497 base pairs) with the mouse genome revealed high sequence similarity (72%) between the orthologous human and mouse STAR18 (SEQ ID NO:18) regions. It also uncovered high similarity (73%) in the region extending for 488 base pairs immediately leftwards of the Sau3AI site that defines the left end of the cloned region (FIG. 21). Outside these regions the sequence similarity between human and mouse DNA drops below 60%.
As indicated in FIG. 19, both the human STAR18 (SEQ ID NO:18) and the mouse STAR18 elements confer survival on zeocin to host cells expressing the lexA-HP1 repressor protein. The original 497 base pair STAR18 clone and its mouse ortholog both confer the ability to grow (FIG. 19, a and d). The adjacent 488 base pair regions of high similarity from both genomes also confer the ability to grow, and in fact their growth phenotype is more vigorous than that of the original STAR18 clone (FIG. 19, b and e). When the entire region of sequence similarity was tested, these DNAs from both mouse and human confer growth, and the growth phenotype is more vigorous than the two sub-fragments (FIG. 19, c and f). These results demonstrate that the STAR activity of human STAR18 (SEQ ID NO:18) is conserved in its ortholog from mouse. The high sequence conservation between these orthologous regions is particularly noteworthy because they are not protein-coding sequences, leading to the conclusion that they have some regulatory function that has prevented their evolutionary divergence through mutation.
This analysis demonstrates that cloned STAR elements identified by the original screening program may in some cases represent partial STAR elements, and that analysis of the genomic DNA in which they are embedded can identify sequences with stronger STAR activity.
Example 13
STAR Elements Contain Characteristic DNA Sequence Motifs
STAR elements are isolated on the basis of their anti-repression phenotype with respect to transgene expression. This anti-repression phenotype reflects underlying biochemical processes that regulate chromatin formation which are associated with the STAR elements. These processes are typically sequence-specific and result from protein binding or DNA structure. This suggests that STAR elements will share DNA sequence similarity. Identification of sequence similarity among STAR elements will provide sequence motifs that are characteristic of the elements that have already been identified by functional screens and tests. The sequence motifs will also be useful to recognize and claim new STAR elements whose functions conform to the claims of this patent. The functions include improved yield and stability of transgenes expressed in eukaryotic host cells.
Other benefits of identifying sequence motifs that characterize STAR elements include: (1) provision of search motifs for prediction and identification of new STAR elements in genome databases, (2) provision of a rationale for modification of the elements, and (3) provision of information for functional analysis of STAR activity. Using bio-informatics, sequence similarities among STAR elements have been identified; the results are presented in this example.
Bio-informatic and Statistical Background. Regulatory DNA elements typically function via interaction with sequence-specific DNA-binding proteins. Bio-informatic analysis of DNA elements such as STAR elements whose regulatory properties have been identified, but whose interacting proteins are unknown, requires a statistical approach for identification of sequence motifs. This can be achieved by a method that detects short DNA sequence patterns that are over-represented in a set of regulatory DNA elements (e.g., the STAR elements) compared to a reference sequence (e.g., the complete human genome). The method determines the number of observed and expected occurrences of the patterns in each regulatory element. The number of expected occurrences is calculated from the number of observed occurrences of each pattern in the reference sequence.
The DNA sequence patterns can be oligonucleotides of a given length, e.g., six base pairs. In the simplest analysis, for a 6 base pair oligonucleotide (hexamer) composed of the four nucleotides (A, C, G, and T) there are 46=4096 distinct oligonucleotides (all combinations from AAAAAA to TTTTTT, SEQ ID NOS:121 and 122, respectively). If the regulatory and reference sequences were completely random and had equal proportions of the A, C, G, and T nucleotides, then the expected frequency of each hexamer would be 1/4096 ( ̃0.00024). However, the actual frequency of each hexamer in the reference sequence is typically different than this due to biases in the content of G:C base pairs, etc. Therefore, the frequency of each oligonucleotide in the reference sequence is determined empirically by counting, to create a “frequency table” for the patterns.
The pattern frequency table of the reference sequence is then used to calculate the expected frequency of occurrence of each pattern in the regulatory element set. The expected frequencies are compared with the observed frequencies of occurrence of the patterns. Patterns that are “over-represented” in the set are identified; for example, if the hexamer ACGTGA (SEQ ID NO:123) is expected to occur five times in 20 kilobase pairs of sequence, but is observed to occur 15 times, then it is three-fold over-represented. Ten of the 15 occurrences of that hexameric sequence pattern would not be expected in the regulatory elements if the elements had the same hexamer composition as the entire genome. Once the over-represented patterns are identified, a statistical test is applied to determine whether their over-representation is significant, or may be due to chance. For this test, a significance index, “sig,” is calculated for each pattern. The significance index is derived from the probability of occurrence of each pattern, which is estimated by a binomial distribution. The probability takes into account the number of possible patterns (4096 for hexamers). The highest sig values correspond to the most overrepresented oligonucleotides (van Helden et al., 1998). In practical terms, oligonucleotides with sig ≧0 are considered as over-represented. A pattern with sig ≧0 is likely to be over-represented due to chance once (=100) in the set of regulatory element sequences. However, at sig ≧1 a pattern is expected to be over-represented once in ten (=101) sequence sets, sig ≧2 once in 100 (=102) sequence sets, etc.
The patterns that are significantly over-represented in the regulatory element set are used to develop a model for classification and prediction of regulatory element sequences. This employs Discriminant Analysis, a so-called “supervised” method of statistical classification known to one of ordinary skill in the art (Huberty, 1994). In Discriminant Analysis, sets of known or classified items (e.g., STAR elements) are used to “train” a model to recognize those items on the basis of specific variables (e.g., sequence patterns such as hexamers). The trained model is then used to predict whether other items should be classified as belonging to the set of known items (e.g., is a DNA sequence STAR element). In this example, the known items in the training set are STAR elements (positive training set). They are contrasted with sequences that are randomly selected from the genome (negative training set) which have the same-length as the STAR elements. Discriminant Analysis establishes criteria for discriminating positives from negatives based on a set of variables that distinguish the positives; in this example, the variables are the significantly over-represented patterns (e.g., hexamers).
When the number of over-represented patterns is high compared to the size of the training set, the model could become biased due to over-training. Over-training is circumvented by applying a forward stepwise selection of variables (Huberty, 1994). The goal of Stepwise Discriminant Analysis is to select the minimum number of variables that provides maximum discrimination between the positives and negatives. The model is trained by evaluating variables one-by-one for their ability to properly classify the items in the positive and negative training sets. This is done until addition of new variables to the model does not significantly increase the model's predictive power (i.e., until the classification error rate is minimized). This optimized model is then used for testing, in order to predict whether “new” items are positives or negatives (Huberty, 1994).
It is inherent in classification statistics that for complex items such as DNA sequences, some elements of the positive training set will be classified as negatives (false negatives), and some members of the negative training set will be classified as positives (false positives). When a trained model is applied to testing new items, the same types of misclassifications are expected to occur.
In the bio-informatic method described here, the first step, pattern frequency analysis, reduces a large set of sequence patterns (e.g., all 4096 hexamers) to a smaller set of significantly over-represented patterns (e.g., 100 hexamers); in the second step, Stepwise Discriminant Analysis reduces the set of over-represented patterns to the subset of those patterns that have maximal discriminative power (e.g., 5-10 hexamers). Therefore, this approach provides simple and robust criteria for identifying regulatory DNA elements such as STAR elements.
DNA-binding proteins can be distinguished on the basis of the type of binding site they occupy. Some recognize contiguous sequences; for this type of protein, patterns that are oligonucleotides of length 6 base pairs (hexamers) are fruitful for bio-informatic analysis (van Helden et al., 1998). Other proteins bind to sequence dyads: contact is made between pairs of highly conserved trinucleotides separated by a non-conserved region of fixed width (van Helden et al., 2000). In order to identify sequences in STAR elements that may be bound by dyad-binding proteins, frequency analysis was also conducted for this type of pattern, where the spacing between the two trinucleotides was varied from 0 to 20 (i.e., XXXN{0-20}XXX where X's are specific nucleotides composing the trinucleotides, and N's are random nucleotides from 0 to 20 base pairs in length). The results of dyad frequency analysis are also used for Linear Discriminant Analysis as described above.
Materials and Methods
Using the genetic screen described herein and in EP 01202581.3, sixty-six (66) STAR elements were initially isolated from human genomic DNA and characterized in detail (Table 6). The screen was performed on gene libraries constructed by Sau3AI digestion of human genomic DNA, either purified from placenta (Clontech 6550-1) or carried in bacterial/P1 (BAC/PAC) artificial chromosomes. The BAC/PAC clones contain genomic DNA from regions of chromosome 1 (clones RP1154H19 and RP3328E19), from the HOX cluster of homeotic genes (clones RP1167F23, RP1170019, and RP11387A1), or from human chromosome 22 (Research Genetics 96010-22). The DNAs were size-fractionated, and the 0.5-2 kb size fraction was ligated into BamHI-digested pSelect vector, by standard techniques (Sambrook et al., 1989). pSelect plasmids containing human genomic DNA that conferred resistance to zeocin at low doxycycline concentrations were isolated and propagated in Escherichia coli. The screens that yielded the STAR elements of Table 6 have assayed approximately 1-2% of the human genome.
The human genomic DNA inserts in these 66 plasmids were sequenced by the dideoxy method (Sanger et al., 1977) using a Beckman CEQ2000 automated DNA sequencer, using the manufacturer's instructions. Briefly, DNA was purified from E. coli using QIAprep® Spin Miniprep and Plasmid Midi Kits (QIAGEN® 27106 and 12145, respectively). Cycle sequencing was carried out using custom oligonucleotides corresponding to the pSelect vector (primers D89 and D95, Table 1), in the presence of dye terminators (CEQTM Dye Terminator Cycle Sequencing Kit, Beckman 608000). Assembled STAR DNA sequences were located in the human genome (database builds August and December 2001) using BLAT (Basic Local Alignment Tool (Kent, 2002); WorldWideWeb.genome.ucsc.edu/cgi-bin/hgGateway; Table 6). In aggregate, the combined STAR sequences comprise 85.6 kilobase pairs, with an average length of 1.3 kilobase pairs.
Sequence motifs that distinguish STAR elements within human genomic DNA were identified by bio-informatic analysis using a two-step procedure, as follows (see FIG. 20 for a schematic diagram). The analysis has two input datasets: (1) the DNA sequences of the STAR elements (STAR1-STAR65 (SEQ ID NOS:1-65) were used; Table 6); and (2) the DNA sequence of the human genome (except for chromosome 1, which was not feasible to include due to its large size; for dyad analysis a random subset of human genomic DNA sequence ( ̃27 Mb) was used).
Pattern Frequency Analysis. The first step in the analysis uses RSA-Tools software (Regulatory Sequence Analysis Tools; WorldWideWeb.ucmb.ulb.ac.be/bioinformatics/rsa-tools/; references (van Helden et al., 1998, van Helden et al., 2000, van Helden et al., 2000)) to determine the following information: (1) the frequencies of all dyads and hexameric oligonucleotides in the human genome; (2) the frequencies of the oligonucleotides and dyads in the 65 STAR elements; and (3) the significance indices of those oligonucleotides and dyads that are over-represented in the STAR elements compared to the genome. A control analysis was done with 65 sequences that were selected at random from the human genome (i.e., from 2689×103 kilobase pairs) that match the length of the STAR elements of Table 6.
Discriminant Analysis. The over-represented oligonucleotides and dyads were used to train models for prediction of STAR elements by Linear Discriminant Analysis (Huberty, 1994). A pre-selection of variables was performed by selecting the 50 patterns with the highest individual discriminatory power from the over-represented oligos or dyads of the frequency analyses. These pre-selected variables were then used for model training in a Stepwise Linear Discriminant Analysis to select the most discriminant combination of variables (Huberty, 1994). Variable selection was based on minimizing the classification error rate (percentage of false negative classifications). In addition, the expected error rate was estimated by applying the same discriminant approach to the control set of random sequences (minimizing the percentage of false positive classifications).
The predictive models from the training phase of Discriminant Analysis were tested in two ways. First, the STAR elements and random sequences that were used to generate the model (the training sets) were classified. Second, sequences in a collection of 19 candidate STAR elements (recently cloned by zeocin selection as described above) were classified. These candidate STAR elements are listed in Table 9 (SEQ ID NOS:67-84).
Results
Pattern frequency analysis was performed with RSA-Tools on 65 STAR elements, using the human genome as the reference sequence. One hundred sixty-six (166) hexameric oligonucleotides were found to be over-represented in the set of STAR elements (sig ≧0) compared to the entire genome (Table 4). The most significantly over-represented oligonucleotide, CCCCAC (SEQ ID NO:391), occurs 107 times among the 65 STAR elements, but is expected to occur only 49 times. It has a significance coefficient of 8.76; in other words, the probability that its over-representation is due to random chance is 1/108.76, i.e., less than one in 500 million.
Ninety-five of the oligonucleotides have a significance coefficient greater than one, and are, therefore, highly over-represented in the STAR elements. Among the over-represented oligonucleotides, their observed and expected occurrences, respectively, range from six and one (for oligo 163, CGCGAA (SEQ ID NO:380), sig=0.02) to 133 and 95 (for oligo 120, CCCAGG (SEQ ID NO:337), sig=0.49). The differences in expected occurrences reflect factors such as the G:C content of the human genome. Therefore, the differences among the oligonucleotides in their number of occurrences is less important than their over-representation; for example, oligo 2 (CAGCGG (SEQ ID NO:386)) is 36/9=4-fold over-represented, which has a probability of being due to random chance of one in fifty million (sig=7.75).
Table 4 also presents the number of STAR elements in which each over-represented oligonucleotide is found. For example, the most significant oligonucleotide, oligo 1 (CCCCAC (SEQ ID NO:391)), occurs 107 times, but is found in only 51 STARs, i.e., on average it occurs as two copies per STAR. The least abundant oligonucleotide, number 166 (AATCGG (SEQ ID NO:383)), occurs on average as a single copy per STAR (thirteen occurrences on eleven STARs); single-copy oligonucleotides occur frequently, especially for the lower-abundance oligos. At the other extreme, oligo 4 (CAGCCC (SEQ ID NO:568)) occurs on average three times in those STARs in which it is found (37 STARs). The most widespread oligonucleotide is number 120 (CCCAGG (SEQ ID NO:337)), which occurs on 58 STARs (on average twice per STAR), and the least widespread oligonucleotide is number 114 (CGTCGC (SEQ ID NO:331)), which occurs on only 6 STARs (and on average only once per STAR).
Results of dyad frequency analysis are given in Table 5. Seven hundred thirty (730) dyads were found to be over-represented in the set of STAR elements (sig ≧0) compared to the reference sequence. The most significantly over-represented dyad, CCCN{2}CGG (SEQ ID NO:384), occurs 36 times among the 65 STAR elements, but is expected to occur only seven times. It has a significance coefficient of 9.31; in other words, the probability that its over-representation is due to chance is 1/109.31, i.e., less than one in two billion.
Three hundred ninety-seven of the dyads have a significance coefficient greater than one, and are, therefore, highly over-represented in the STAR elements. Among the over-represented dyads, their observed and expected occurrences, respectively, range from nine and one (for five dyads (numbers 380 (SEQ ID NO:763), 435 (SEQ ID NO:818), 493 (SEQ ID NO:876), 640 (SEQ ID NO:1023), and 665 (SEQ ID NO:1048))) to 118 and 63 (for number 30 (AGGN{2}GGG) (SEQ ID NO:413), sig=4.44).
The oligonucleotides and dyads found to be over-represented in STAR elements by pattern frequency analysis were tested for their discriminative power by Linear Discriminant Analysis. Discriminant models were trained by step-wise selection of the best combination among the 50 most discriminant oligonucleotide (Table 4) or dyad (Table 5) patterns. The models achieved optimal error rates after incorporation of four (dyad) or five variables. The discriminative variables from oligo analysis are numbers 11 (SEQ ID NO:228), 30 (SEQ ID NO:247), 94 (SEQ ID NO:311), 122 (SEQ ID NO:339), and 160 (SEQ ID NO:377)(Table 4); those from dyad analysis are numbers 73 (SEQ ID NO:456), 194 (SEQ ID NO:577), 419 (SEQ ID NO:802), and 497 (SEQ ID NO:880) (Table 5).
The discriminant models were then used to classify the 65 STAR elements in the training set and their associated random sequences. The model using oligonucleotide variables classifies 46 of the 65 STAR elements as STAR elements (true positives); the dyad model classifies 49 of the STAR elements as true positives. In combination, the models classify 59 of the 65 STAR elements as STAR elements (91%; FIG. 21). The false positive rates (random sequences classified as STARs) were seven for the dyad model, eight for the oligonucleotide model, and 13 for the combined predictions of the two models (20%). The STAR elements of Table 6 that were not classified as STARs by LDA are STARs 7 (SEQ ID NO:7), 22 (SEQ ID NO:22), 35 (SEQ ID NO:35), 44 (SEQ ID NO:44), 46 (SEQ ID NO:46), and 65 (SEQ ID NO:65). These elements display stabilizing anti-repressor activity in functional assays, so the fact that they are not classified as STARs by LDA suggests that they represent another class (or classes) of STAR elements.
The models were then used to classify the 19 candidate STAR elements in the testing set listed in Table 9. The dyad model classifies 12 of these candidate STARs as STAR elements, and the oligonucleotide model classifies 14 as STARs. The combined number of the candidates that are classified as STAR elements is 15 (79%). This is a lower rate of classification than obtained with the training set of 65 STARs; this is expected for two reasons. First, the discriminant models were trained with the 65 STARs of Table 6, and discriminative variables based on this training set may be less well represented in the testing set. Second, the candidate STAR sequences in the testing set have not yet been fully characterized in terms of in vivo function, and may include elements with only weak anti-repression properties.
This analysis demonstrates the power of a statistical approach to bio-informatic classification of STAR elements. The STAR sequences contain a number of dyad and hexameric oligonucleotide patterns that are significantly over-represented in comparison with the human genome as a whole. These patterns may represent binding sites for proteins that confer STAR activity; in any case they form a set of sequence motifs that can be used to recognize STAR element sequences.
Using these patterns to recognize STAR elements by Discriminant Analysis, a high proportion of the elements obtained by the genetic screen of the invention are in fact classified as STARs. This reflects underlying sequence and functional similarities among these elements. An important aspect of the method described here (pattern frequency analysis followed by Discriminant Analysis) is that it can be reiterated; for example, by including the 19 candidate STAR elements of Table 9 with the 66 STAR elements of Table 6 into one training set, an improved discriminant model can be trained. This improved model can then be used to classify other candidate regulatory elements as STARs. Large-scale in vivo screening of genomic sequences using the method of the invention, combined with reiteration of the bio-informatic analysis, will provide a means of discriminating STAR elements that asymptotically approaches 100% recognition and prediction of elements as the genome is screened in its entirety. These stringent and comprehensive predictions of STAR function will ensure that all human STAR elements are recognized, and are available for use in improving transgene expression.
Example 14
Cloning and characterization of STAR elements from Arabidopsis thaliana 
Transgene silencing occurs in transgenic plants at both the transcriptional and post-transcriptional levels (Meyer, 2000, Vance & Vaucheret, 2001). In either case, the desired result of transgene expression can be compromised by silencing; the low expression and instability of the transgene results in poor expression of desirable traits (e.g., pest resistance) or low yields of recombinant proteins. It also results in poor predictability: the proportion of transgenic plants that express the transgene at biotechnologically useful levels is low, which necessitates laborious and expensive screening of transformed individuals for those with beneficial expression characteristics. This example describes the isolation of STAR elements from the genome of the dicot plant Arabidopsis thaliana for use in preventing transcriptional transgene silencing in transgenic plants. Arabidopsis was chosen for this example because it is a well-studied model organism: it has a compact genome, it is amenable to genetic and recombinant DNA manipulations, and its genome has been sequenced (Bevan et al., 2001, Initiative, 2000, Meinke et al., 1998).
Materials and Methods:
Genomic DNA was isolated from Arabidopsis thaliana ecotype Columbia as described (Stam et al., 1998) and partially digested with MboI. The digested DNA was size-fractionated to 0.5-2 kilobase pairs by agarose gel electrophoresis and purification from the gel (QIAquick® Gel Extraction Kit, QIAGEN® 28706), followed by ligation into the pSelect vector (supra). Transfection into the U-2 OS/Tet-Off/LexA-HP1 cell line and selection for zeocin resistance at low doxycycline concentration was performed as described (supra). Plasmids were isolated from zeocin resistant colonies and re-transfected into the U-2 OS/Tet-Off/LexA-HP1 cell line.
Sequencing of Arabidopsis genomic DNA fragments that conferred zeocin resistance upon re-transfection was performed as described (supra). The DNA sequences were compared to the sequence of the Arabidopsis genome by BLAST analysis ((Altschul et al., 1990); URL WorldWideWeb.ncbi.nlm.nih.gov/blast/Blast).
STAR activity was tested further by measuring mRNA levels for the hygromycin-and zeocin-resistance genes in recombinant host cells by reverse transcription PCR(RT-PCR). Cells of the U-2 OS/Tet-Off/lexA-HP1 cell line were transfected with pSelect plasmids containing Arabidopsis STAR elements, the Drosophila scs element, or containing no insert (supra). These were cultivated on hygromycin for two weeks at high doxycycline concentration, then the doxycycline concentration was lowered to 0.1 ng/ml to induce the lexA-HP1 repressor protein. After ten days, total RNA was isolated by the RNeasy mini kit (QIAGEN® 74104) as described by the manufacturer. First-strand cDNA synthesis was carried out using the RevertAidTM First Strand cDNA Synthesis kit (MBI Fermentas 1622) using oligo(dT)18 primer as described by the manufacturer. An aliquot of the cDNA was used as the template in a PCR reaction using primers D58 (SEQ ID NO:151) and D80 (SEQ ID NO:154) (for the zeocin marker), and D70 (SEQ ID NO:152) and D71 (SEQ ID NO:153) (for the hygromycin marker), and Taq DNA polymerase (Promega M2661). The reaction conditions were 15-20 cycles of 94° C. for one minute, 54° C. for one minute, and 72° C. for 90 seconds. These conditions result in a linear relationship between input RNA and PCR product DNA. The PCR products were resolved by agarose gel electrophoresis, and the zeocin and hygromycin bands were detected by Southern blotting as described (Sambrook et al., 1989), using PCR products produced as above with purified pSelect plasmid as template. The ratio of the zeocin and hygromycin signals corresponds to the normalized expression level of the zeocin gene.
Results
The library of Arabidopsis genomic DNA in the pSelect vector comprised 69,000 primary clones in E. coli, 80% of which carried inserts. The average insert size was approximately 1000 base pairs; the library, therefore, represents approximately 40% of the Arabidopsis genome.
A portion of this library (representing approximately 16% of the Arabidopsis genome) was transfected into the U-2 OS/Tet-Off/LexA-HP1 cell line. Hygromycin selection was imposed to isolate transfectants, which resulted in 27,000 surviving colonies. These were then subjected to zeocin selection at low doxycycline concentration. Putative STAR-containing plasmids from 56 zeocin-resistant colonies were rescued into E. coli and re-transfected into U-2 OS/Tet-Off/LexA-HP1 cells. Forty-four of these plasmids (79% of the plasmids tested) conferred zeocin resistance on the host cells at low doxycycline concentrations, demonstrating that the plasmids carried STAR elements. This indicates that the pSelect screen in human U-2 OS cells is highly efficient at detection of STAR elements from plant genomic DNA.
The DNA sequences of these 44 candidate STAR elements were determined. Thirty-five of them were identified as single loci in the database of Arabidopsis nuclear genomic sequence (Table 10; SEQ ID NO:85-SEQ ID NO:119). Four others were identified as coming from the chloroplast genome, four were chimeras of DNA fragments from two loci, and one was not found in the Arabidopsis genome database.
The strength of the cloned Arabidopsis STAR elements was tested by assessing their ability to prevent transcriptional repression of the zeocin-resistance gene, using an RT-PCR assay. As a control for RNA input among the samples, the transcript levels of the hygromycin-resistance gene for each STAR transfection were assessed too. This analysis has been performed for 12 of the Arabidopsis STAR elements. The results (FIG. 22) demonstrate that the Arabidopsis STAR elements are superior to the Drosophila scs element (positive control) and the empty vector (“SV40”; negative control) in their ability to protect the zeocin-resistance gene from transcriptional repression. In particular, STAR-A28 (SEQ ID NO:112) and STAR-A30 (SEQ ID NO:114) enable 2-fold higher levels of zeocin-resistance gene expression than the scs element (normalized to the internal control of hygromycin-resistance gene mRNA) when the lexA-HP1 repressor is expressed.
These results demonstrate that the method of the invention can be successfully applied to recovery of STAR elements from genomes of other species than human. Its successful application to STAR elements from a plant genome is particularly significant because it demonstrates the wide taxonomic range over which the method of the invention is applicable, and because plants are an important target of biotechnological development.
Example 15
STAR-Shielded Genes that Reside on Multiple Vectors are Expressed Simultaneously in Cho Cells
STAR elements function to block the effect of transcriptional repression influences on transgene expression units. One of the benefits of STAR elements for heterologous protein production is the increased predictability of finding high-expressing primary recombinant host cells. This feature allows for the simultaneous expression of different genes that reside on multiple, distinct vectors. In this example, we use two different STAR7-shielded (SEQ ID NO:7) genes, GFP and RED, which are located on two different vectors. When these two vectors are transfected simultaneously to Chinese hamster ovary (CHO) cells, both are expressed, whereas the corresponding, but unprotected GFP and RED genes, show hardly such simultaneous expression.
Material and Methods
The STAR7 element (SEQ ID NO:7) is tested in the ppGIZ-STAR7 and ppRIP-STAR7 vectors (FIG. 23). The construction of the pPlug&Play (ppGIZ and ppRIP) vectors is described below. Plasmid pGFP (Clontech 6010-1) is modified by insertion of a linker at the BsiWI site to yield pGFP-link. The linker (made by annealing oligonucleotides 5′GTACGGATATCAGATCTTTAATTAAG3′ (SEQ ID NO:124) and 5′GTACCTTAATTAAAGATCTGATATCC3′ (SEQ ID NO:125)) introduces sites for the PacI, BglII, and EcoRV restriction endonucleases. This creates the multiple cloning site MCSII for insertion of STAR elements. Then primers (5′GATCAGATCTGGCGCGCCATTTAAATCG TCTCGCGCGTTTCGGTGATGACGG3′ (SEQ ID NO:126)) and (5′AGGCGGATCCGA ATGTATTTAGAAAAATAAACAAA TAGGGG3′ (SEQ ID NO:127)) are used to amplify a region of 0.37 kb from pGFP, which is inserted into the BglII site of pIRES (Clontech 6028-1) to yield pIRES-stuf. This introduces sites for the AscI and SwaI restriction endonucleases at MCSI, and acts as a “stuffer fragment” to avoid potential interference between STAR elements and adjacent promoters. pIRES-stuf is digested with BglII and FspI to liberate a DNA fragment composed of the stuffer fragment, the CMV promoter, the IRES element (flanked by multiple cloning sites MCS A and MCS B), and the SV40 polyadenylation signal. This fragment is ligated with the vector backbone of pGFP-link produced by digestion with BamHI and StuI, to yield pIRES-link.
The open reading frames of the zeocin-resistance gene are inserted into the BamHI/NotI sites of MCS B in pIRES-link as follows: the zeocin-resistance ORF is amplified by PCR with primers 5′GATCGGATCCTTCGAAATGGCCAAGTTGACCAGTGC3′ (SEQ ID NO:128) and 5′AGGCGCGGCCGCAATTCTCAGTCCTGCTCCTC3′ (SEQ ID NO:129) from plasmid pEM7/zeo, digested with BamHI and NotI, and ligated with BamHI/NotI-digested pIRES-link to yield pIRES-link-zeo. The GFP reporter ORF is introduced into pIRES-link-zeo by amplification of phr-GFP-1 with primers 5′GATCGAATTCTCGCGAATGGTGAGCAAGC AGATCCTGAAG3′ (SEQ ID NO:130) and 5′AGGCGAATTCACCGGTGTTTAAACTTAC ACCCACTCGTGCAGGCTGCCCAGG3′ (SEQ ID NO:131), and insertion of the EcoRI-digested GFP cassette into the EcoRI site in MCS A of the pIRES-link-zeo plasmid. This creates the ppGIZ (for ppGFP-IRES-zeo). 5′ STAR7 (SEQ ID NO:7) is cloned into the SalI site and 3′ STAR7 (SEQ ID NO:7) is cloned into the PacI site.
The puromycin-resistance ORF is amplified by PCR with primers 5′GATCGGATCCTTCGAAATGACCGAGTACAAGCCCACG3′ (SEQ ID NO:132) and 5′AGGCGCGGCCGCTCAGGCACCGGGCTTGCGGGTC3′ (SEQ ID NO:133) from plasmid pBabe-Puro (Morgenstern & Land, 1990), digested with BamHI and NotI, and ligated with BamHI/NotI-digested pIRES-link to yield pIRES-link-puro. The RED gene is amplified by PCR with primers 5′GATCTCTAGATCGCGAATGGCCTCCTCCGAGAACGTCATC3′ (SEQ ID NO:134) and 5′AGGCACGCGTTCGCGACTACAGGAACAGGTGGTGGCG3′ (SEQ ID NO:135) from plasmid pDsRed2 (Clontech 6943-1), digested with XbaI and MluI and ligated to NheI-MluI digested pIRES-link-puro to yield ppRIP (for ppRED-IRES-puro). 5′ STAR7 (SEQ ID NO:7) is cloned into the SalI site and 3′ STAR7 (SEQ ID NO:7) is cloned into the PacI site.
Transfection and culture of CHO cells
The Chinese Hamster Ovary cell line CHO-K1 (ATCC CCL-61) is cultured in HAMS-F12 medium +10% Fetal Calf Serum containing 2 mM glutamine, 100 U/ml penicillin, and 100 micrograms/ml streptomycin at 37° C./5% CO2. Cells are transfected with the plasmids using LipofectamineTM 2000 (Invitrogen) as described by the manufacturer. Briefly, cells are seeded to culture vessels and grown overnight to 70-90% confluence. Lipofectamine reagent is combined with plasmid DNA at a ratio of 7.5 microliters per 3 microgram (e.g., for a 10 cm Petri dish, 20 micrograms DNA and 120 microliters Lipofectamine) and added after 30 minutes incubation at 25° C. to the cells. After six hours incubation, the transfection mixture is replaced with fresh medium, and the transfected cells are incubated further. After overnight cultivation, cells are trypsinized and seeded into fresh petri dishes with fresh medium with zeocin added to a concentration of 100 μg/ml and the cells are cultured further. When individual colonies become visible (approximately ten days after transfection) medium is removed and replaced with fresh medium (puromycin).
Individual colonies are isolated and transferred to 24-well plates in medium with zeocin. Expression of the GFP and RED reporter genes is assessed approximately three weeks after transfection.
One tested construct consists of a monocistronic gene with the GFP gene, an IRES and the Zeocin resistance gene under control of the CMV promoter, but either with or without STAR7 element (SEQ ID NO:7) to flank the entire construct (FIG. 23). The other construct consists of a monocistronic gene with the RED gene, an IRES and the puromycin resistance gene under control of the CMV promoter, but either with or without STAR7 element (SEQ ID NO:7) to flank the entire construct (FIG. 23).
The constructs are transfected to CHO-K1 cells. Stable colonies that are resistant for both zeocin and puromycin are expanded before the GFP and RED signals are determined on a XL-MCL Beckman Coulter flow cytometer. The percentage of cells in one colony that are double positive for both GFP and RED signals is taken as measure for simultaneous expression of both proteins and this is plotted in FIG. 23.
Results
FIG. 23 shows that simultaneous expression in independent zeocin and puromycin resistant CHO colonies of GFP and a RED reporter genes that are flanked by a STAR element results in a higher number of cells that express both GFP and RED proteins, as compared to the control vectors without STAR7 element (SEQ ID NO:7). The STAR7 element (SEQ ID NO:7), therefore, conveys a higher degree of predictability of transgene expression in CHO cells. In the STAR-less colonies at most nine out of 20 colonies contain double GFP/RED positive cells. The percentage of double positive cells ranges between 10 and 40%. The remaining 11 out of 20 colonies have less than 10% GFP/RED positive cells. In contrast, in 19 out of 20 colonies that contain the STAR-shielded GFP and RED genes, the percentage GFP/RED double positive cells ranges between 25 and 75%. In 15 out of these 19 double positive colonies the percentage GFP/RED double positive cells is higher than 40%. This result shows that it is more likely that simultaneous expression of two genes is achieved when these genes are flanked with STAR elements.
Example 16
Expression of a Functional Antibody from Two Separate Plasmids is More Easily Obtained when Star Elements Flank the Genes Encoding the Heavy and Light Chains
Due to the ability of STAR elements to convey higher predictability to protein expression two genes can be expressed simultaneously from distinct vectors. This is shown in Example 15 for two reporter genes, GFP and RED. Now the simultaneous expression of a light and a heavy antibody chain is tested. In Example 16, STAR7-shielded light and heavy antibody cDNAs that reside on distinct vectors are simultaneously transfected to Chinese hamster ovary cells. This results in the production of functional antibody, indicating that both heavy and light chains are expressed simultaneously. In contrast, the simultaneous transfection of unprotected light and heavy antibody cDNAs shows hardly any expression of functional antibody.
Materials and Methods
The tested constructs are the same as described in Example 15, except that the GFP gene is replaced by the gene encoding the light chain of the RING1 antibody (Hamer et al., 2002) and the RED gene is replaced by the gene encoding the heavy chain of the RING1 antibody. The light chain is amplified from the RING1 hybridoma (Hamer et al., 2002) by RT-PCR using the primers 5′CAAGAATTCAATGGATTTTCAAGTGCAG3′ (SEQ ID NO:136) and 5′CAAGCGGCCGCTTTGTCTCTAACACTCATTCC3′ (SEQ ID NO:137). The PCR product is cloned into pcDNA3 after restriction digestion with EcoRI and NotI and sequenced to detect potential frame shifts in the sequence. The cDNA is excised with EcoRI and NotI, blunted and cloned in ppGIZ plasmid. The heavy chain is amplified from the RING1 hybridoma (Hamer et al., 2002) by RT-PCR using the primers 5′ACAGAATTCTTACCATGGATTTTGGGCTG3′ (SEQ ID NO:138) and 5′ACAGCGGCCGCTCATTTACCAGGAGAGTGGG3′ (SEQ ID NO:139). The PCR product is cloned into pcDNA3 after restriction digestion with EcoRI and NotI and sequenced to detect potential frame shifts in the sequence. The cDNA is excised with EcoRI and NotI, blunted and cloned in ppRIP plasmid.
Results
CHO colonies are simultaneously transfected with the RING1 Light Chain (LC) and RING1 Heavy Chain (HC) cDNAs that reside on two distinct vectors. The Light Chain is coupled to the zeocin resistance gene through an IRES, the Heavy Chain is coupled to the puromycin resistance gene through an IRES. FIG. 24 shows that simultaneous transfection to CHO cells of the heavy and light chain encoding cDNAs results in the establishment of independent zeocin and puromycin resistant colonies. When the constructs are flanked by the STAR7 element (SEQ ID NO:7), this results in a higher production of functional RING1 antibody, as compared to the control vectors without STAR7 element (SEQ ID NO:7). The STAR7 element (SEQ ID NO:7), therefore, conveys a higher degree of predictability of antibody expression in CHO cells.
In the STAR-less colonies only one out of 12 colonies express detectable antibody. In contrast, seven out of twelve colonies that contain the STAR-shielded Light and Heavy Chain genes produce functional RING1 antibody that detects the RING1 antigen in an ELISA assay. Significantly, all these seven colonies produce higher levels of RING1 antibody than the highest control colony (arbitrarily set at 100%). This result shows that it is more likely that simultaneous expression of two genes encoding two antibody chains is achieved when these genes are flanked with STAR elements.








TABLE 1







Oligonucleotides used for polymerase



chain reactions (PCR primers) or DNA


mutagenesis (SEQ ID NOS:140-207).









SEQ ID NO:
Number
Sequence













140
C65
AACAAGCTTGATATCAGATCTGCTAGCTTGGT





CGAGCTGATACTTCCC


 


141
C66
AAACTCGAGCGGCCGCGAATTCGTCGACTTTA




CCACTCCCTATCAGTGATAGAG


 


142
C67
AAACCGCGGCATGGAAGACGCCAAAAACATAA




AGAAAGG


 


143
C68
TATGGATCCTAGAATTACACGGCGATCTTTCC


 


144
C81
AAACCATGGCCGAGTACAAGCCCACGGTGCGC




C


 


145
C82
AAATCTAGATCAGGCACCGGGCTTGCGGGTCA




TGC


 


146
C85
CATTTCCCCGAAAAGTGCCACC


 


147
D30
TCACTGCTAGCGAGTGGTAAACTC


 


148
D41
GAAGTCGACGAGGCAGGCAGAAGTATGC


 


149
D42
GAGCCGCGGTTTAGTTCCTCACCTTGTCG


 


150
D51
TCTGGAAGCTTTGCTGAAGAAAC


 


151
D58
CCAAGTTGACCAGTGCC


 


152
D70
TACAAGCCAACCACGGCCT


 


153
D71
CGGAAGTGCTTGACATTGGG


 


154
D80
GTTCGTGGACACGACCTCCG


 


155
D89
GGGCAAGATGTCGTAGTCAGG


 


156
D90
AGGCCCATGGTCACCTCCATCGCTACTGTG


 


157
D91
CTAATCACTCACTGTGTAAT


 


158
D93
AATTACAGGCGCGCC


 


159
D94
AATTGGCGCGCCTGT


 


160
D95
TGCTTTGCATACTTCTGCCTGCCTC


 


161
E12
TAGGGGGGATCCAAATGTTC


 


162
E13
CCTAAAAGAAGATCTTTAGC


 


163
E14
AAGTGTTGGATCCACTTTGG


 


164
E15
TTTGAAGATCTACCAAATGG


 


165
E16
GTTCGGGATCCACCTGGCCG


 


166
E17
TAGGCAAGATCTTGGCCCTC


 


167
E18
CCTCTCTAGGGATCCGACCC


 


168
E19
CTAGAGAGATCTTCCAGTAT


 


169
E20
AGAGTTCCGGATCCGCCTGG


 


170
E21
CCAGGCAGACTCGGAACTCT


 


171
E22
TGGTGAAACCGGATCCCTAC


 


172
E23
AGGTCAGGAGATCTAGACCA


 


173
E25
CCATTTTCGCTTCCTTAGCTCC


 


174
E42
CGATGTAACCCACTCGTGCACC


 


175
E57
AGAGATCTAGGATAATTTCG


 


176
E84
GATCTCTAGAATGGCCAAGCCTTTGTCTCAAG


 


177
E85
AGGCGCGGCCGCTTAGCCCTCCCACACATAAC




CAGAG


 


178
E87
AGGCACGCGTTCATGTCTGCTCGAAGCGGCC


 


179
E92
AGGCGCTAGCACGCGTTCTACTCTTTTCCTAC




TCTG


 


180
E93
GATCAAGCTTACGCGTCTAAAGGCATTTTATA




TAG


 


181
E94
AGGCGCTAGCACGCGTTCAGAGTTAGTGATCC




AGG


 


182
E95
GATCAAGCTTACGCGTCAGTAAAGGTTTCGTA




TGG


 


183
E96
AGGCGCTAGCACGCGTTCTACTCTTTCATTAC




TCTG


 


184
E97
CGAGGAAGCTGGAGAAGGAGAAGCTG


 


185
E98
CAAGGGCCGCAGCTTACACATGTTC


 


186
E99
GATCACTAGTATGGCCAAGTTGACCAGTGC


 


187
E100
AGGCGCGGCCGCAATTCTCAGTCCTGCTCCTC


 


188
F11
GATCGCTAGCAATCGCGACTTCGCCCACCATG




C


 


189
F14
GATCGAATTCTCGCGACTTCGCCCACCATGC


 


190
F15
AGGCGAATTCACCGGTGTTTAAACTCATGTCT




GCTCGAAGCGGCCGG


 


191
F16
GATCGAATTCTCGCGAATGGTGAGCAAGCAGA




TCCTGAAG


 


192
F17
AGGCGAATTCACCGGTGTTTAAACTTACACCC




ACTCGTGCAGGCTGCCCAGG


 


193
F18
GATCGGATCCTTCGAAATGGCCAAGTTGACCA




GTGC


 


194
F19
GATCGGATCCTTCGAAATGATTGAACAAGATG




GATTGC


 


195
F20
AGGCGCGGCCGCTCAGAAGAACTCGTCAAGAA




GGCG


 


196
F21
GATCGGATCCTTCGAAATGACCGAGTACAAGC




CCACG


 


197
F22
AGGCGCGGCCGCTCAGGCACCGGGCTTGCGGG




TC


 


198
F23
GATCAGATCTGGCGCGCCATTTAAATCGTCTC




GCGCGTTTCGGTGATGACGG


 


199
F24
AGGCGGATCCGAATGTATTTAGAAAAATAAAC




AAATAGGGG


 


200
F25
GTACGGATATCAGATCTTTAATTAAG


 


201
F26
GTACCTTAATTAAAGATCTGATATCC


 


202
F32
GATCGAGGTACCGGTGTGT


 


203
F33
GATCACACACCGGTACCTC


 


204
F34
CGGAGGTACCGGTGTGT


 


205
F35
CGACACACCGGTACCTC


 


206
F44
TGAGAGGTACCGGTGTGT


 


207
F45
TCAACACACCGGTACCTC
















TABLE 2







STAR elements and two-step selection increase


the predictability of transgene expression











without STAR
fold improvement
with STAR














(carry out first





antibiotic selection)


Number colonies1
~100
10-fold
~1000


High producers


percent
5%
 3-fold
15% 


number
5

150


(characterize 20
(20% of

(2% of


colonies)
population)

population)


High producers
1
 3-fold2
3


Low producers
19

17


(carry out second


antibiotic selection,


killing low producers)


Survivors to
5
30-fold3
150


characterize





1Colonies per microgram plasmid DNA.


2Manifesting the three-fold improvement due to the presence of STARs in the percent of high producers in the original population of colonies resistant to the first antibiotic.


3Manifesting the arithmetic product of the fold improvement in the number of colonies and the increased percentage of high producers due to the presence of STARs.














TABLE 3





Sequences of various STAR elements



(SEQ ID NOS:208-217)
















STAR3 forward









(SEQ ID NO:208)









ACGTNCTAAGNAAACCATTATTATCATGACATTAACCTATAAAAATAGGC



GTATCACGAGGCCCTTTCGTCTTCACTCGAGCGGCCAGCTTGGATCTCGA


GTACTGAAATAGGAGTAAATCTGAAGAGCAAATAAGATGAGCCAGAAAAC


CATGAAAAGAACAGGGACTACCAGTTGATTCCACAAGGACATTCCCAAGG


TGAGAAGGCCATATACCTCCACTACCTGAACCAATTCTCTGTATGCAGAT


TTAGCAAGGTTATAAGGTAGCAAAAGATTAGACCCAAGAAAATAGAGAAC


TTCCAATCCAGTAAAAATCATAGCAAATTTATTGATGATAACAATTGTCT


CCAAAGGAACCAGGCAGAGTCGTGCTAGCAGAGGAAGCACGTGAGCTGAA


AACAGCCAAATCTGCTTTGTTTTCATGACACAGGAGCATAAAGTACACAC


CACCAACTGACCTATTAAGGCTGTGGTAAACCGATTCATAGAGAGAGGTT


CTAAATACATTGGTCCCTCATAGGCAAACCGCAGTTCACTCCGAACGTAG


TCCCTGGAAATTTGATGTCCAGNATAGAAAAGCANAGCAGNCNNNNNNTA


TANATNNNGNTGANCCANATGNTNNCTGNNC


 


STAR3 reverse








(SEQ ID NO:209)









GAGCTAGCGGCGCGCCAAGCTTGGATCCCGCCCCGCCCCCTCCGCCCTCG



AGCCCCGCCCCTTGCCCTAGAGGCCCTGCCGAGGGGCGGGGCCTGTCCCT


CCTCCCCTTTCCCCCGCCCCCTACCGTCACGCTCAGGGGCAGCCTGACCC


CGAGCGGCCCCGCGGTGACCCTCGCGCAGAGGCCTGTGGGAGGGGCGTCG


CAAGCCCCTGAATCCCCCCCCGTCTGTTCCCCCCTCCCGCCCAGTCTCCT


CCCCCTGGGAACGCGCGGGGTGGGTGACAGACCTGGCTGCGCGCCACCGC


CACCGCGCCTGCCGGGGGCGCTGCCGCTGCCTGAGAAACTGCGGCTGCCG


CCTGGAGGAGGTGCCGTCGCCTCCGCCACCGCTGCCGCCGCCGCCAGGGG


TAGGAGCTAAGCCGCCGCCATTTTGTGTCCCCCTGTTGTTGTCGTTGACA


TGAATCCGACATGACACTGATTACAGCCCAATGGAGTCTCATTAAACCCG


AGTCGCGGTCCCGCCCCGCCGCTGCTCCATTGGAGGAGACCAAAGACACT


TAAGGCCACCCGTTGGCCTACGGGTCTGTCTGTCACCCACTCACTAACCA


CTCTGCAGCCCATTGGGGCAGGTTCCTGCCGGTCATNTCGCTTCCAATAA


ACACACCCCTTCGACCCCATNATTCCCCCCCTTCGGGAACCACCCCCGGG


GGAGGGGTCCACTGGNCAATACCAATTNAANAGAACCGCTNGGGTCCGCC


TNTTTNCGGGCNCCCTATTGGGTT


 


STAR4 forward








(SEQ ID NO:210)









GGGGAGGATTCTTTTGGCTGCTGAGTTGAGATTAGGTTGAGGGTAGTGAA



GGTAAAGGCAGTGAGACCACGTAGGGGTCATTGCAGTAATCCAGGCTGGA


GATGATGGTGGTTCAGTTGGAATAGCAGTGCATGTGCTGTAACAACCTCA


GCTGGGAAGCAGTATATGTGGCGTTATGACCTCAGCTGGAACAGCAATGC


ATGTGGTGGTGTAATGACCCCAGCTGGGTAGGGTGCATGTGATGGAACAA


CCTCAGCTGGGTAGCAGTGTACTTGATAAAATGTTGGCATACTCTACATT


TGTTATGAGGGTAGTGCCATTAAATTTCTCCACAAATTGGTTGTCACGTA


TGAGTGAAAAGAGGAAGTGATGGAAGACTTCAGTGCTTTTGGCCTGAATA


AATAGAAGACGTCATTTTCAGTAATGGAGACAGGGAAGACTAANGNAGGG


TGGATTCAGTAGAGCAGGTGTTCAGTTTTGAATATGATGAACTCTGAGAG


AGGAAAAACTTTTTCTACCTCTTAGTTTTTGNGNCTGGACTTAANATTAA


AGGACATANGACNGAGANCAGACCAAATNTGCGANGTTTTTATATTTTAC


TTGCNGAGGGAATTTNCAAGAAAAAGAAGACCCAANANCCATTGGTCAAA


ACTATNTGCCTTTTAANAAAAAGANAATTACAATGGANANANAAGTGTTG


NCTNGGCAAAAATTGGG


 


STAR4 reverse








(SEQ ID NO:211)









GGATTNGAGCTAGCGGCGCGCCAAGCTTGGATCTTAGAAGGACAGAGTGG



GGCATGGAAATGCACCACCAGGGCAGTGCAGCTTGGTCACTGCCAGCTCC


NCTCATGGGCAGAGGGCTGGCCTCTTGCAGCCGACCAGGCACTGAGCGCC


ATCCCAGGGCCCTCGCCAGCCCTCAGCAGGGCCAGGACACACAAGCCTTT


GACTTCCTCCTGTCACTGCTGCTGCCATTCCTGTTTTGTGGTCATCACTC


CTTCCCTGTCCTCAGACTGCCCAGCACTCAAGGATGTCCTGTGGTGGCAT


CAGACCATATGCCCCTGAANAGGAGTGAGTTGGTGTTTTTTGCCGCGCCC


ANAGAGCTGCTGTCCCCTGAAAGATGCAAGTGGGAATGATGATGNTCACC


ATCNTCTGACACCAAGCCCTTTGGATAGAGGCCCCAACAGTGAGGATGGG


GCTGCACTGCATTGCCAAGGCAACTCTGTNNTGACTGCTACANGACANTC


CCAGGACCTGNGAAGNNCTATANATNTGATGCNAGGCACCT


 


STAR6 forward








(SEQ ID NO:212)









CCACCACAGACATCCCCTCTGGCCTCCTGAGTGGTTTCTTCAGCACAGCT



TCCAGAGCCAAATTAAACGTTCACTCTATGTCTATAGACAAAAAGGGTTT


TGACTAAACTCTGTGTTTTAGAGAGGGAGTTAAATGCTGTTAACTTTTTA


GGGGTGGGCGAGAGGAATGACAAATAACAACTTGTCTGAATGTTTTACAT


TTCTCCCCACTGCCTCAAGAAGGTTCACAACGAGGTCATCCATGATAAGG


AGTAAGACCTCCCAGCCGGACTGTCCCTCGGCCCCCAGAGGACACTCCAC


AGAGATATGCTAACTGGACTTGGAGACTGGCTCACACTCCAGAGAAAAGC


ATGGAGCACGAGCGCACAGAGCANGGGCCAAGGTCCCAGGGACNGAATGT


CTAGGAGGGAGATTGGGGTGAGGGTANTCTGATGCAATTACTGNGCAGCT


CAACATTCAAGGGAGGGGAAGAAAGAAACNGTCCCTGTAAGTAAGTTGTN


CANCAGAGATGGTAAGCTCCAAATTTNAACTTTGGCTGCTGGAAAGTTTN


NGGGCCNANANAANAAACANAAANATTTGAGGTTTANACCCACTAACCCN


TATNANTANTTATTAATACCCCTAATTANACCTTGGATANCCTTAAAATA


TCNTNTNAAACGGAACCCTCNTTCCCNTTTNNAAATNNNAAAGGCCATTN


NGNNCNAGTAAAAATCTNNNTTAAGNNNTGGGCCCNAACAAACNTNTTCC


NAGACACNTTTTTTNTCCNGGNATTTNTAATTTATTTCTAANCC


 


STAR6 reverse








(SEQ ID NO:213)









ATCGTGTCCTTTCCAGGGACATGGATGAAGCTGGAAGCCATCATCCTCAG



CAAACTAACACAGGAACAGAAAACCAAATACCACATGTTCTCACTCATAA


GTGGGAGCTGAACAGTGAGAACACATGGACACAGGGAGGGGAACATCACA


CACCAAGGCCTGTCTGGTGTGGGGAGGGGAGGGAGAGCATCAGGACAAAT


AGCTAATGCATGTGGGGCTTAAACCTAGATGACGGGTTGATAGGTGCAGC


AATCCACTATGGACACATATACCTATGTAACAACCCNACCTTNTTGACAT


GTATCCCAGAACTTAAAGGAAAATAAAAATTAAAAAAAATTNCCCTGGAA


TAAAAAAGAGTGTGGACTTTGGTGAGATN


 


STAR8 forward








(SEQ ID NO:214)









GGATCACCTCGAAGAGAGTCTAACGTCCGTAGGAACGCTCTCGGGTTCAC



AAGGATTGACCGAACCCCAGGATACGTCGCTCTCCATCTGAGGCTTGNTC


CAAATGGCCCTCCACTATTCCAGGCACGTGGGTGTCTCCCCTAACTCTCC


CTGCTCTCCTGAGCCCATGCTGCCTATCACCCATCGGTGCAGGTCCTTTC


TGAANAGCTCGGGTGGATTCTCTCCATCCCACTTCCTTTCCCAAGAAAGA


AGCCACCGTTCCAAGACACCCAATGGGACATTCCCNTTCCACCTCCTTNT


CNAAAGTTNGCCCAGGTGTTCNTAACAGGTTAGGGAGAGAANCCCCCAGG


TTTNAGTTNCAAGGCATAGGACGCTGGCTTGAACACACACACACNCTC


 


STAR8 reverse








(SEQ ID NO:215)









GGATCCCGACTCTGCACCGCAAACTCTACGGCGCCCTGCAGGACGGCGGC



CTCCTGCCGCTTGGACGCCAGNCAGGAGCTCCCCGGCAGCAGCAGAGCAG


AAAGAAGGATGGCCCCGCCCCACTTCGCCTCCCGGCGGTCTCCCTCCCGC


CGGCTCACGGACATAGATGGCTGCCTAGCTCCGGAAGCCTAGCTCTTGTT


CCGGGCATCCTAAGGAAGACACGGTTTTTCCTCCCGGGGCCTCACCACAT


CTGGGACTTTGACGACTCGGACCTCTCTCCATTGAATGGTTGCGCGTTCT


CTGGGAAAG


 


STAR18 forward








(SEQ ID NO:216)









TGGATCCTGCCGCTCGCGTCTTAGTGTTTCTCCCTCAAGACTTTCCTTCT



GTTTTGTTGTCTTGTGCAGTATTTTACAGCCCCTCTTGTGTTTTTCTTTA


TTTCTCGTACACACACGCAGTTTTAAGGGTGATGTGTGTATAATTAAAAG


GACCCTTGGCCCATACTTTCCTAATTCTTTAGGGACTGGGATTGGGTTTG


ACTGAAATATGTTTTGGTGGGGATGGGACGGTGGACTTCCATTCTCCCTA


AACTGGAGTTTTGGTCGGTAATCAAAACTAAAAGAAACCTCTGGGAGACT


GGAAACCTGATTGGAGCACTGAGGAACAAGGGAATGAAAAGGCAGACTCT


CTGAACGTTTGATGAAATGGACTCTTGTGAAAATTAACAGTGAATATTCA


CTGTTGCACTGTACGAAGTCTCTGAAATGTAATTAAAAGTTTTTATTGAG


CCCCCGAGCTTTGGCTTGCGCGTATTTTTCCGGTCGCGGACATCCCACCG


CGCAGAGCCTCGCCTCCCCGCTGNCCTCAGCTCCGATGACTTCCCCGCCC


CCGCCCTGCTCGGTGACAGACGTTCTACTGCTTCCAATCGGAGGCACCCT


TCGCGG


 


STAR18 reverse








(SEQ ID NO:217)









TGGATCCTGCCGCTCGCGTCTTAGTGTTTCTCCCTCAAGACTTTCCTTCT



GTTTTGTTGTCTTGTGCAGTATTTTACAGCCCCTCTTGTGTTTTTCTTTA


TTTCTCGTACACACACGCAGTTTTAAGGGTGATGTGTGTATAATTAAAAG


GACCCTTGGCCCATACTTTCCTAATTCTTTAGGGACTGGGATTGGGTTTG


ACTGAAATATGTTTTGGTGGGGATGGGACGGTGGACTTCCATTCTCCCTA


AACTGGAGTTTTGGTCGGTAATCAAAACTAAAAGAAACCTCTGGGAGACT


GGAAACCTGATTGGAGCACTGAGGAACAAGGGAATGAAAAGGCAGACTCT


CTGAACGTTTGATGAAATGGACTCTTGTGAAAATTAACAGTGAATATTCA


CTGTTGCACTGTACGAAGTCTCTGAAATGTAATTAAAAGTTTTTATTGAG


CCCCCGAGCTTTGGC

















TABLE 4







Oligonucleotide patterns (6 base pairs)



over-represented in STAR elements.



















Number









of



Oligonucleotide
Observed
Expected
Significance
matching


Number
sequence
occurrences
occurrences
coefficient
STARs
SEQ ID NO:

















1
CCCCAC
107
49
8.76
51
218



 


2
CAGCGG
36
9
7.75
23
219


 


3
GGCCCC
74
31
7.21
34
220


 


4
CAGCCC
103
50
7.18
37
221


 


5
GCCCCC
70
29
6.97
34
222


 


6
CGGGGC
40
12
6.95
18
223


 


7
CCCCGC
43
13
6.79
22
224


 


8
CGGCAG
35
9
6.64
18
225


 


9
AGCCCC
83
38
6.54
40
226


 


10
CCAGGG
107
54
6.52
43
227


 


11
GGACCC *
58
23
6.04
35
228


 


12
GCGGAC
20
3
5.94
14
229


 


13
CCAGCG
34
10
5.9
24
230


 


14
GCAGCC
92
45
5.84
43
231


 


15
CCGGCA
28
7
5.61
16
232


 


16
AGCGGC
27
7
5.45
17
233


 


17
CAGGGG
86
43
5.09
43
234


 


18
CCGCCC
43
15
5.02
18
235


 


19
CCCCCG
35
11
4.91
20
236


 


20
GCCGCC
34
10
4.88
18
237


 


21
GCCGGC
22
5
4.7
16
238


 


22
CGGACC
19
4
4.68
14
239


 


23
CGCCCC
35
11
4.64
19
240


 


24
CGCCAG
28
8
4.31
19
241


 


25
CGCAGC
29
8
4.29
20
242


 


26
CAGCCG
32
10
4
24
243


 


27
CCCACG
33
11
3.97
26
244


 


28
GCTGCC
78
40
3.9
43
245


 


29
CCCTCC
106
60
3.87
48
246


 


30
CCCTGC *
92
50
3.83
42
247


 


31
CACCCC
77
40
3.75
40
248


 


32
GCGCCA
30
10
3.58
23
249


 


33
AGGGGC
70
35
3.55
34
250


 


34
GAGGGC
66
32
3.5
40
251


 


35
GCGAAC
14
2
3.37
13
252


 


36
CCGGCG
17
4
3.33
12
253


 


37
AGCCGG
34
12
3.29
25
254


 


38
GGAGCC
67
34
3.27
40
255


 


39
CCCCAG
103
60
3.23
51
256


 


40
CCGCTC
24
7
3.19
19
257


 


41
CCCCTC
81
44
3.19
43
258


 


42
CACCGC
33
12
3.14
22
259


 


43
CTGCCC
96
55
3.01
42
260


 


44
GGGCCA
68
35
2.99
39
261


 


45
CGCTGC
28
9
2.88
22
262


 


46
CAGCGC
25
8
2.77
19
263


 


47
CGGCCC
28
10
2.73
19
264


 


48
CCGCCG
19
5
2.56
9
265


 


49
CCCCGG
30
11
2.41
17
266


 


50
AGCCGC
23
7
2.34
17
267


 


51
GCACCC
55
27
2.31
38
268


 


52
AGGACC
54
27
2.22
33
269


 


53
AGGGCG
24
8
2.2
18
270


 


54
CAGGGC
81
47
2.18
42
271


 


55
CCCGCC
45
21
2.15
20
272


 


56
GCCAGC
66
36
2.09
39
273


 


57
AGCGCC
21
6
2.09
18
274


 


58
AGGCCC
64
34
2.08
32
275


 


59
CCCACC
101
62
2.05
54
276


 


60
CGCTCA
21
6
2.03
17
277


 


61
AACGCG
9
1
1.96
9
278


 


62
GCGGCA
21
7
1.92
14
279


 


63
AGGTCC
49
24
1.87
36
280


 


64
CCGTCA
19
6
1.78
14
281


 


65
CAGAGG
107
68
1.77
47
282


 


66
CCCGAG
33
14
1.77
22
283


 


67
CCGAGG
36
16
1.76
25
284


 


68
CGCGGA
11
2
1.75
8
285


 


69
CCACCC
87
53
1.71
45
286


 


70
CCTCGC
23
8
1.71
20
287


 


71
CAAGCC
59
32
1.69
40
288


 


72
TCCGCA
18
5
1.68
17
289


 


73
CGCCGC
18
5
1.67
9
290


 


74
GGGAAC
55
29
1.63
39
291


 


75
CCAGAG
93
58
1.57
49
292


 


76
CGTTCC
19
6
1.53
16
293


 


77
CGAGGA
23
8
1.5
19
294


 


78
GGGACC
48
24
1.48
31
295


 


79
CCGCGA
10
2
1.48
8
296


 


80
CCTGCG
24
9
1.45
17
297


 


81
CTGCGC
23
8
1.32
14
298


 


82
GACCCC
47
24
1.31
33
299


 


83
GCTCCA
66
38
1.25
39
300


 


84
CGCCAC
33
15
1.19
21
301


 


85
GCGGGA
23
9
1.17
18
302


 


86
CTGCGA
18
6
1.15
15
303


 


87
CTGCTC
80
49
1.14
50
304


 


88
CAGACG
23
9
1.13
19
305


 


89
CGAGAG
21
8
1.09
17
306


 


90
CGGTGC
18
6
1.06
16
307


 


91
CTCCCC
84
53
1.05
47
308


 


92
GCGGCC
22
8
1.04
14
309


 


93
CGGCGC
14
4
1.04
13
310


 


94
AAGCCC *
60
34
1.03
42
311


 


95
CCGCAG
24
9
1.03
17
312


 


96
GCCCAC
59
34
0.95
35
313


 


97
CACCCA
92
60
0.93
49
314


 


98
GCGCCC
27
11
0.93
18
315


 


99
ACCGGC
15
4
0.92
13
316


 


100
CTCGCA
16
5
0.89
14
317


 


101
ACGCTC
16
5
0.88
12
318


 


102
CTGGAC
58
33
0.88
32
319


 


103
GCCCCA
67
40
0.87
38
320


 


104
ACCGTC
15
4
0.86
11
321


 


105
CCCTCG
21
8
0.8
18
322


 


106
AGCCCG
22
8
0.79
14
323


 


107
ACCCGA
16
5
0.78
13
324


 


108
AGCAGC
79
50
0.75
41
325


 


109
ACCGCG
14
4
0.69
7
326


 


110
CGAGGC
29
13
0.69
24
327


 


111
AGCTGC
70
43
0.64
36
328


 


112
GGGGAC
49
27
0.64
34
329


 


113
CCGCAA
16
5
0.64
12
330


 


114
CGTCGC
8
1
0.62
6
331


 


115
CGTGAC
17
6
0.57
15
332


 


116
CGCCCA
33
16
0.56
22
333


 


117
CTCTGC
97
65
0.54
47
334


 


118
AGCGGG
21
8
0.52
17
335


 


119
ACCGCT
15
5
0.5
11
336


 


120
CCCAGG
133
95
0.49
58
337


 


121
CCCTCA
71
45
0.49
39
338


 


122
CCCCCA *
77
49
0.49
42
339


 


123
GGCGAA
16
5
0.48
14
340


 


124
CGGCTC
29
13
0.47
19
341


 


125
CTCGCC
20
8
0.46
17
342


 


126
CGGAGA
20
8
0.45
14
343


 


127
TCCCCA
95
64
0.43
52
344


 


128
GACACC
44
24
0.42
33
345


 


129
CTCCGA
17
6
0.42
13
346


 


130
CTCGTC
17
6
0.42
14
347


 


131
CGACCA
13
4
0.39
11
348


 


132
ATGACG
17
6
0.37
12
349


 


133
CCATCG
17
6
0.37
13
350


 


134
AGGGGA
78
51
0.36
44
351


 


135
GCTGCA
77
50
0.35
43
352


 


136
ACCCCA
76
49
0.33
40
353


 


137
CGGAGC
21
9
0.33
16
354


 


138
CCTCCG
28
13
0.32
19
355


 


139
CGGGAC
16
6
0.3
10
356


 


140
CCTGGA
88
59
0.3
45
357


 


141
AGGCGA
18
7
0.29
17
358


 


142
ACCCCT
54
32
0.28
36
359


 


143
GCTCCC
56
34
0.27
36
360


 


144
CGTCAC
16
6
0.27
15
361


 


145
AGCGCA
16
6
0.26
11
362


 


146
GAAGCC
62
38
0.25
39
363


 


147
GAGGCC
79
52
0.22
42
364


 


148
ACCCTC
54
32
0.22
33
365


 


149
CCCGGC
37
20
0.21
21
366


 


150
CGAGAA
20
8
0.2
17
367


 


151
CCACCG
29
14
0.18
20
368


 


152
ACTTCG
16
6
0.17
14
369


 


153
GATGAC
48
28
0.17
35
370


 


154
ACGAGG
23
10
0.16
18
371


 


155
CCGGAG
20
8
0.15
18
372


 


156
ACCCAC
60
37
0.12
41
373


 


157
CTGGGC
105
74
0.11
50
374


 


158
CCACGG
23
10
0.09
19
375


 


159
CGGTCC
13
4
0.09
12
376


 


160
AGCACC *
54
33
0.09
40
377


 


161
ACACCC
53
32
0.08
38
378


 


162
AGGGCC
54
33
0.08
30
379


 


163
CGCGAA
6
1
0.02
6
380


 


164
GAGCCC
58
36
0.02
36
381


 


165
CTGAGC
71
46
0.02
45
382


 


166
AATCGG
13
4
0.02
11
383





The patterns are ranked according to significance coefficient. These were determined using RSA-Tools with the sequence of the human genome as reference. Patterns that comprise the most discriminant variables in Linear Discriminant Analysis are indicated with an asterisk (SEQ ID NOS:218-383).













TABLE 5







Dyad patterns over-represented in STAR elements.















Observed
Expected
Significance




Number
Dyad sequence
occurrences
occurrences
coefficient
SEQ ID NO:
















1
CCCN{2}CGG
36
7
9.31
384



 


2
CCGN{6}CCC
40
10
7.3
385


 


3
CAGN{0}CGG
36
8
7.13
386


 


4
CGCN{15}CCC
34
8
6.88
387


 


5
CGGN{9}GCC
33
7
6.82
388


 


6
CCCN{9}CGC
35
8
6.72
389


 


7
CCCN{1}GCG
34
8
6.64
390


 


8
CCCN{0}CAC
103
48
6.61
391


 


9
AGCN{16}CCG
29
6
5.96
392


 


10
CCCN{4}CGC
34
8
5.8
393


 


11
CGCN{13}GGA
26
5
5.77
394


 


12
GCGN{16}CCC
30
7
5.74
395


 


13
CGCN{5}GCA
25
5
5.49
396


 


14
CCCN{14}CCC
101
49
5.43
397


 


15
CTGN{4}CGC
34
9
5.41
398


 


16
CCAN{12}GCG
28
6
5.37
399


 


17
CGGN{11}CAG
36
10
5.25
400


 


18
CCCN{5}GCC
75
33
4.87
401


 


19
GCCN{0}CCC
64
26
4.81
402


 


20
CGCN{4}GAC
19
3
4.78
403


 


21
CGGN{0}CAG
33
9
4.76
404


 


22
CCCN{3}CGC
32
8
4.67
405


 


23
CGCN{1}GAC
20
3
4.58
406


 


24
GCGN{2}GCC
29
7
4.54
407


 


25
CCCN{4}GCC
76
34
4.53
408


 


26
CCCN{1}CCC
103
52
4.53
409


 


27
CCGN{13}CAG
33
9
4.5
410


 


28
GCCN{4}GGA
64
27
4.48
411


 


29
CCGN{3}GGA
26
6
4.46
412


 


30
AGGN{2}GGG
118
63
4.44
413


 


31
CACN{5}GCG
22
4
4.42
414


 


32
CGCN{17}CCA
27
6
4.39
415


 


33
CCCN{9}GGC
69
30
4.38
416


 


34
CCTN{5}GCG
28
7
4.37
417


 


35
GCGN{0}GAC
19
3
4.32
418


 


36
GCCN{0}GGC
40
7
4.28
419


 


37
GCGN{2}CCC
26
6
4.27
420


 


38
CCGN{11}CCC
32
9
4.17
421


 


39
CCCN{8}TCG
23
5
4.12
422


 


40
CCGN{17}GCC
30
8
4.12
423


 


41
GGGN{5}GGA
101
52
4.11
424


 


42
GGCN{6}GGA
71
32
4.1
425


 


43
CCAN{4}CCC
96
48
4.1
426


 


44
CCTN{14}CCG
32
9
4.09
427


 


45
GACN{12}GGC
45
16
4.07
428


 


46
CGCN{13}CCC
30
8
4.04
429


 


47
CAGN{16}CCC
92
46
4.02
430


 


48
AGCN{10}GGG
75
35
3.94
431


 


49
CGGN{13}GGC
30
8
3.93
432


 


50
CGGN{1}GCC
30
8
3.92
433


 


51
AGCN{0}GGC
26
6
3.9
434


 


52
CCCN{16}GGC
64
28
3.89
435


 


53
GCTN{19}CCC
67
29
3.87
436


 


54
CCCN{16}GGG
88
31
3.81
437


 


55
CCCN{9}CGG
30
8
3.77
438


 


56
CCCN{10}CGG
30
8
3.76
439


 


57
CCAN{0}GCG
32
9
3.75
440


 


58
GCCN{17}CGC
26
6
3.74
441


 


59
CCTN{6}CGC
27
7
3.73
442


 


60
GGAN{1}CCC
63
27
3.71
443


 


61
CGCN{18}CAC
24
5
3.7
444


 


62
CGCN{20}CCG
21
4
3.69
445


 


63
CCGN{0}GCA
26
6
3.69
446


 


64
CGCN{20}CCC
28
7
3.69
447


 


65
AGCN{15}CCC
67
30
3.65
448


 


66
CCTN{7}GGC
69
31
3.63
449


 


67
GCCN{5}CGC
32
9
3.61
450


 


68
GCCN{14}CGC
28
7
3.59
451


 


69
CAGN{11}CCC
89
45
3.58
452


 


70
GGGN{16}GAC
53
21
3.57
453


 


71
CCCN{15}GCG
25
6
3.57
454


 


72
CCCN{0}CGC
37
12
3.54
455


 


73
CCCN{16}AGC *
67
30
3.54
456


 


74
AGGN{9}GGG
96
50
3.52
457


 


75
CGCN{12}CTC
28
7
3.46
458


 


76
CACN{8}CGC
23
5
3.43
459


 


77
CCAN{7}CCG
31
9
3.42
460


 


78
CGGN{1}GCA
25
6
3.41
461


 


79
CGCN{14}CCC
29
8
3.4
462


 


80
AGCN{0}CCC
76
36
3.4
463


 


81
CGCN{13}GTC
18
3
3.37
464


 


82
GCGN{3}GCA
26
7
3.35
465


 


83
CGGN{0}GGC
34
11
3.35
466


 


84
GCCN{14}CCC
68
31
3.33
467


 


85
ACCN{7}CGC
21
4
3.32
468


 


86
AGGN{7}CGG
33
10
3.31
469


 


87
CCCN{16}CGA
22
5
3.3
470


 


88
CGCN{6}CAG
31
9
3.29
471


 


89
CAGN{11}GCG
29
8
3.29
472


 


90
CCGN{12}CCG
19
4
3.26
473


 


91
CGCN{18}CAG
27
7
3.24
474


 


92
CAGN{1}GGG
80
39
3.21
475


 


93
CGCN{0}CCC
32
10
3.2
476


 


94
GCGN{18}GCC
26
7
3.18
477


 


95
CGGN{15}GGC
27
7
3.15
478


 


96
CCCN{15}AGG
72
34
3.14
479


 


97
AGGN{20}GCG
26
7
3.14
480


 


98
CGGN{5}CTC
26
7
3.13
481


 


99
TCCN{17}CGA
23
5
3.12
482


 


100
GCGN{4}CCC
30
9
3.08
483


 


101
CCCN{2}CGC
30
9
3.07
484


 


102
CGTN{3}CAG
28
8
3.06
485


 


103
CCGN{13}GAG
27
7
3.05
486


 


104
CTCN{6}CGC
28
8
3.04
487


 


105
CGCN{4}GAG
21
5
3.03
488


 


106
GCGN{5}GGA
24
6
3.03
489


 


107
CCGN{1}CAG
27
7
3.01
490


 


108
CGCN{11}CCG
18
3
2.99
491


 


109
GCGN{19}CCC
26
7
2.98
492


 


110
CGCN{18}GAA
21
5
2.98
493


 


111
GGGN{19}GGA
78
39
2.95
494


 


112
CCAN{1}CGG
24
6
2.94
495


 


113
CCCN{7}GCG
25
6
2.94
496


 


114
AGGN{10}CCC
84
43
2.92
497


 


115
CCAN{0}GGG
97
52
2.88
498


 


116
CAGN{10}CCC
82
41
2.87
499


 


117
CCGN{18}CCG
19
4
2.86
500


 


118
CCGN{18}GGC
26
7
2.85
501


 


119
CCCN{2}GCG
24
6
2.84
502


 


120
CGCN{1}GGC
25
7
2.83
503


 


121
CCGN{5}GAC
19
4
2.81
504


 


122
GGAN{0}CCC
52
22
2.8
505


 


123
CCCN{1}CCG
29
9
2.78
506


 


124
CCCN{15}ACG
23
6
2.75
507


 


125
AGCN{8}CCC
66
31
2.73
508


 


126
CCCN{3}GGC
60
27
2.71
509


 


127
AGGN{9}CGG
31
10
2.7
510


 


128
CCCN{14}CGC
27
8
2.7
511


 


129
CCGN{0}CCG
19
4
2.7
512


 


130
CGCN{8}AGC
23
6
2.69
513


 


131
CGCN{19}ACC
21
5
2.68
514


 


132
GCGN{17}GAC
17
3
2.66
515


 


133
AGCN{1}GCG
24
6
2.63
516


 


134
CCGN{11}GGC
31
10
2.63
517


 


135
CGGN{4}AGA
26
7
2.63
518


 


136
CGCN{14}CCG
17
3
2.62
519


 


137
CCTN{20}GCG
24
6
2.62
520


 


138
CCAN{10}CGC
26
7
2.61
521


 


139
CCCN{20}CAC
69
33
2.6
522


 


140
CCGN{11}GCC
27
8
2.6
523


 


141
CGCN{18}CCC
26
7
2.59
524


 


142
CGGN{15}CGC
16
3
2.57
525


 


143
CGCN{16}GCC
24
6
2.55
526


 


144
CGCN{20}GGC
23
6
2.54
527


 


145
CGCN{19}CCG
18
4
2.52
528


 


146
CGGN{10}CCA
28
8
2.51
529


 


147
CGCN{17}CCC
26
7
2.51
530


 


148
CGCN{11}ACA
23
6
2.51
531


 


149
CGGN{0}ACC
17
3
2.5
532


 


150
GCGN{10}GCC
24
6
2.49
533


 


151
GCGN{8}GAC
17
3
2.49
534


 


152
CCCN{15}GGG
84
32
2.44
535


 


153
CGGN{16}GGC
27
8
2.44
536


 


154
CGCN{16}CCA
23
6
2.42
537


 


155
GCCN{3}CCC
73
36
2.4
538


 


156
CAGN{4}GGG
94
51
2.4
539


 


157
CCCN{6}GCG
23
6
2.38
540


 


158
CCGN{16}CGC
17
3
2.38
541


 


159
CCCN{17}GCA
61
28
2.37
542


 


160
CGCN{13}TCC
24
6
2.37
543


 


161
GCCN{1}CGC
29
9
2.36
544


 


162
CCGN{19}GAG
26
7
2.35
545


 


163
GGGN{10}GGA
89
48
2.35
546


 


164
CAGN{5}CCG
32
11
2.35
547


 


165
CGCN{3}AGA
19
4
2.32
548


 


166
GCCN{0}GCC
29
9
2.32
549


 


167
CCCN{8}GGC
61
28
2.31
550


 


168
CCTN{6}GCG
22
6
2.29
551


 


169
GACN{6}CCC
48
20
2.29
552


 


170
CGGN{1}CCC
26
8
2.27
553


 


171
CCCN{15}CCG
30
10
2.27
554


 


172
CAGN{9}CCC
84
44
2.26
555


 


173
CGGN{10}GGC
27
8
2.26
556


 


174
CGAN{10}ACG
10
1
2.26
557


 


175
GCGN{3}TCC
21
5
2.26
558


 


176
CCCN{3}GCC
75
38
2.24
559


 


177
GCGN{1}ACC
17
3
2.24
560


 


178
CCGN{9}AGG
27
8
2.23
561


 


179
CGCN{16}CAG
26
8
2.23
562


 


180
GGCN{0}CCC
62
29
2.22
563


 


181
AGGN{12}CCG
26
8
2.19
564


 


182
CCGN{0}GCG
16
3
2.19
565


 


183
CCGN{2}GCC
30
10
2.18
566


 


184
CCGN{11}GTC
19
4
2.17
567


 


185
CAGN{0}CCC
88
47
2.17
568


 


186
CCCN{5}CCG
32
11
2.17
569


 


187
GCCN{20}CCC
66
32
2.15
570


 


188
GACN{2}CGC
18
4
2.14
571


 


189
CGCN{6}CAC
23
6
2.13
572


 


190
AGGN{14}GCG
25
7
2.1
573


 


191
GACN{5}CGC
17
3
2.1
574


 


192
CCTN{9}CCG
29
9
2.1
575


 


193
CCGN{12}GGA
24
7
2.08
576


 


194
GGCN{9}GAC *
44
18
2.08
577


 


195
AGGN{10}GGG
94
52
2.07
578


 


196
CCGN{10}GAG
25
7
2.07
579


 


197
CGCN{6}GGA
20
5
2.06
580


 


198
CGCN{7}AGC
23
6
2.04
581


 


199
CCAN{13}CGG
26
8
2.03
582


 


200
CGGN{6}GGA
25
7
2.03
583


 


201
CGCN{19}GCC
24
7
2.03
584


 


202
CCAN{12}CGC
24
7
2.02
585


 


203
CGGN{1}GGC
41
16
2.02
586


 


204
GCGN{3}CCA
25
7
2.01
587


 


205
AGGN{1}CGC
21
5
2
588


 


206
CTCN{5}CGC
24
7
1.98
589


 


207
CCCN{0}ACG
30
10
1.97
590


 


208
CAGN{17}CCG
29
9
1.96
591


 


209
GGCN{4}CCC
62
30
1.96
592


 


210
AGGN{8}GCG
26
8
1.96
593


 


211
CTGN{1}CCC
88
48
1.94
594


 


212
CCCN{16}CAG
85
46
1.94
595


 


213
CGCN{9}GAC
16
3
1.93
596


 


214
CAGN{6}CCG
29
9
1.92
597


 


215
CGTN{12}CGC
11
1
1.92
598


 


216
CTCN{7}GCC
69
35
1.92
599


 


217
CGCN{19}TCC
22
6
1.92
600


 


218
CCCN{7}GCC
67
33
1.91
601


 


219
CAGN{13}CGG
30
10
1.9
602


 


220
CGCN{1}GCC
27
8
1.9
603


 


221
CGCN{17}CCG
17
4
1.89
604


 


222
AGGN{4}CCC
63
31
1.89
605


 


223
AGCN{10}CGC
21
5
1.89
606


 


224
CCCN{11}CGG
30
10
1.88
607


 


225
CCCN{8}GCC
75
39
1.86
608


 


226
CCGN{1}CGG
22
3
1.86
609


 


227
CCCN{1}ACC
71
36
1.85
610


 


228
CGCN{0}CAG
25
7
1.85
611


 


229
CCGN{19}TGC
23
6
1.82
612


 


230
GCGN{4}CGA
12
2
1.82
613


 


231
CCGN{19}GCC
30
10
1.82
614


 


232
CCAN{10}CCC
85
46
1.81
615


 


233
CAGN{13}GGG
91
51
1.81
616


 


234
AGCN{18}CGG
23
6
1.81
617


 


235
CGAN{8}CGC
11
1
1.81
618


 


236
AGCN{4}CCC
63
31
1.8
619


 


237
GGAN{6}CCC
61
30
1.8
620


 


238
CGGN{13}AAG
23
6
1.8
621


 


239
ACCN{11}CGC
19
5
1.79
622


 


240
CCGN{12}CAG
28
9
1.78
623


 


241
CCCN{12}GGG
76
29
1.77
624


 


242
CACN{17}ACG
22
6
1.76
625


 


243
CAGN{18}CCC
82
44
1.76
626


 


244
CGTN{10}GTC
19
5
1.75
627


 


245
CCCN{13}GCG
23
6
1.75
628


 


246
GCAN{1}CGC
20
5
1.73
629


 


247
AGAN{4}CCG
24
7
1.73
630


 


248
GCGN{10}AGC
22
6
1.72
631


 


249
CGCN{0}GGA
12
2
1.72
632


 


250
CGGN{4}GAC
17
4
1.69
633


 


251
CCCN{12}CGC
26
8
1.68
634


 


252
GCCN{15}CCC
65
33
1.68
635


 


253
GCGN{6}TCC
20
5
1.66
636


 


254
CGGN{3}CAG
33
12
1.65
637


 


255
CCCN{3}CCA
88
49
1.65
638


 


256
AGCN{3}CCC
59
28
1.65
639


 


257
GGGN{16}GCA
65
33
1.65
640


 


258
AGGN{8}CCG
28
9
1.64
641


 


259
CCCN{0}CCG
29
10
1.64
642


 


260
GCGN{5}GAC
16
3
1.64
643


 


261
CCCN{9}ACC
60
29
1.64
644


 


262
CTGN{5}CGC
25
8
1.64
645


 


263
CGCN{14}CTC
23
7
1.64
646


 


264
CGGN{14}GCA
23
7
1.63
647


 


265
CCGN{8}GCC
26
8
1.62
648


 


266
CCGN{7}CAC
23
7
1.62
649


 


267
AGCN{8}GCG
21
6
1.61
650


 


268
CGGN{16}GGA
29
10
1.61
651


 


269
CCAN{12}CCG
26
8
1.61
652


 


270
CGGN{2}CCC
26
8
1.6
653


 


271
CCAN{13}GGG
71
37
1.6
654


 


272
CGGN{15}GCA
21
6
1.6
655


 


273
CGCN{9}GCA
20
5
1.58
656


 


274
CGGN{19}CCA
26
8
1.58
657


 


275
GGGN{15}CGA
20
5
1.57
658


 


276
CCCN{10}CGC
26
8
1.57
659


 


277
CTCN{14}CGC
26
8
1.55
660


 


278
CACN{11}GCG
20
5
1.55
661


 


279
CCGN{2}GGC
24
7
1.55
662


 


280
CTGN{18}CCC
85
47
1.54
663


 


281
GGGN{13}CAC
58
28
1.54
664


 


282
CCTN{15}GGC
62
31
1.54
665


 


283
CCCN{20}CGA
20
5
1.54
666


 


284
CCCN{8}CGA
20
5
1.53
667


 


285
GAGN{7}CCC
61
30
1.53
668


 


286
CGCN{2}CCG
22
6
1.53
669


 


287
CCCN{0}TCC
98
57
1.52
670


 


288
AGCN{0}GCC
21
6
1.52
671


 


289
CCCN{2}TCC
82
45
1.52
672


 


290
CCGN{5}CCC
30
10
1.52
673


 


291
CGCN{13}CGC
16
3
1.51
674


 


292
CCCN{1}CGC
28
9
1.51
675


 


293
GCCN{16}GCA
53
25
1.51
676


 


294
CCCN{16}CCA
84
46
1.5
677


 


295
CCGN{13}CGC
19
5
1.5
678


 


296
CCGN{17}CAG
28
9
1.49
679


 


297
CGGN{18}GGC
26
8
1.49
680


 


298
CCGN{14}AGG
23
7
1.49
681


 


299
CCCN{5}CGG
26
8
1.49
682


 


300
CCCN{6}GGA
58
28
1.49
683


 


301
ACGN{2}CCC
20
5
1.49
684


 


302
CCAN{9}CCG
27
9
1.48
685


 


303
CCCN{19}CCA
78
42
1.48
686


 


304
CAGN{0}GGG
77
41
1.48
687


 


305
AGCN{1}CCC
58
28
1.47
688


 


306
GCGN{7}TCC
27
9
1.46
689


 


307
ACGN{18}CCA
25
8
1.46
690


 


308
GCTN{14}CCC
61
30
1.46
691


 


309
GCGN{14}CCC
23
7
1.46
692


 


310
GCGN{19}AGC
20
5
1.45
693


 


311
CCGN{8}CAG
29
10
1.45
694


 


312
GCGN{6}GCC
22
6
1.45
695


 


313
GCGN{10}GCA
20
5
1.44
696


 


314
CCTN{7}GCC
69
36
1.44
697


 


315
GCCN{13}GCC
54
26
1.42
698


 


316
CCCN{14}GCC
63
32
1.42
699


 


317
CCCN{15}CGG
26
8
1.42
700


 


318
CCAN{13}CGC
23
7
1.42
701


 


319
AGCN{11}GGG
67
35
1.41
702


 


320
GGAN{0}GCC
64
32
1.4
703


 


321
GCCN{3}TCC
61
30
1.4
704


 


322
CCTN{5}GCC
69
36
1.39
705


 


323
CGGN{18}CCC
25
8
1.39
706


 


324
CCTN{3}GGC
59
29
1.38
707


 


325
CCGN{0}CTC
22
6
1.38
708


 


326
AGCN{17}GCG
19
5
1.37
709


 


327
ACGN{14}GGG
20
5
1.37
710


 


328
CGAN{12}GGC
19
5
1.37
711


 


329
CCCN{20}CGC
24
7
1.37
712


 


330
ACGN{12}CTG
24
7
1.36
713


 


331
CCGN{0}CCC
36
14
1.36
714


 


332
CCGN{10}GGA
23
7
1.36
715


 


333
CCCN{3}GCG
21
6
1.36
716


 


334
GCGN{14}CGC
22
3
1.35
717


 


335
CCGN{8}CGC
16
4
1.35
718


 


336
CGCN{10}ACA
22
6
1.34
719


 


337
CCCN{19}CCG
28
10
1.33
720


 


338
CACN{14}CGC
20
5
1.32
721


 


339
GACN{3}GGC
46
21
1.32
722


 


340
GAAN{7}CGC
19
5
1.32
723


 


341
CGCN{16}GGC
21
6
1.31
724


 


342
GGCN{9}CCC
64
33
1.31
725


 


343
CCCN{9}GCC
64
33
1.31
726


 


344
CGCN{0}TGC
26
9
1.3
727


 


345
CCTN{8}GGC
67
35
1.3
728


 


346
CCAN{8}CCC
82
46
1.29
729


 


347
GACN{2}CCC
42
18
1.28
730


 


348
GGCN{1}CCC
54
26
1.27
731


 


349
CGCN{0}AGC
24
7
1.26
732


 


350
AGGN{4}GCG
28
10
1.26
733


 


351
CGGN{6}TCC
22
6
1.25
734


 


352
ACGN{19}GGC
20
5
1.25
735


 


353
CCCN{8}ACG
21
6
1.24
736


 


354
CCCN{18}GCC
62
31
1.24
737


 


355
GCCN{2}CGA
19
5
1.24
738


 


356
CCCN{8}GCG
28
10
1.23
739


 


357
CCCN{0}CTC
76
41
1.23
740


 


358
GCCN{11}CGC
27
9
1.22
741


 


359
AGCN{9}CCC
59
29
1.22
742


 


360
GCTN{0}GCC
71
38
1.21
743


 


361
CGCN{3}CCC
26
9
1.21
744


 


362
CCCN{2}CCC
117
72
1.19
745


 


363
GCCN{9}CGC
23
7
1.19
746


 


364
GCAN{19}CGC
19
5
1.19
747


 


365
CAGN{4}CGG
32
12
1.18
748


 


366
CAGN{2}GGG
80
44
1.17
749


 


367
GCCN{16}CCC
67
35
1.16
750


 


368
GAGN{5}CCC
60
30
1.16
751


 


369
CCTN{16}TCG
20
6
1.16
752


 


370
CCCN{2}GGC
62
32
1.15
753


 


371
GCGN{13}GGA
24
8
1.15
754


 


372
GCCN{17}GGC
66
25
1.15
755


 


373
CCCN{14}GGC
58
29
1.14
756


 


374
AGGN{3}CCG
31
12
1.14
757


 


375
CACN{0}CGC
32
12
1.14
758


 


376
CGGN{18}CAG
28
10
1.14
759


 


377
AGCN{1}GCC
57
28
1.13
760


 


378
CGCN{18}GGC
23
7
1.13
761


 


379
CCCN{}AGG
64
33
1.11
762


 


380
AACN{0}GCG
9
1
1.11
763


 


381
CCCN{10}CCA
88
50
1.09
764


 


382
CGCN{13}GAG
20
6
1.09
765


 


383
CGCN{7}GCC
25
8
1.08
766


 


384
CCCN{9}CCG
28
10
1.07
767


 


385
CGCN{16}CCC
24
8
1.05
768


 


386
GAAN{13}CGC
18
5
1.05
769


 


387
GGCN{3}CCC
49
23
1.03
770


 


388
TCCN{11}CCA
87
50
1.03
771


 


389
CACN{0}CCC
70
38
1.02
772


 


390
CGCN{16}CCG
15
3
1.02
773


 


391
CGGN{15}AGC
21
6
1.02
774


 


392
CCCN{12}GCG
21
6
1.02
775


 


393
CCCN{9}GAG
59
30
1.01
776


 


394
CCGN{20}TCC
24
8
1.01
777


 


395
CGCN{0}CGC
17
4
1.01
778


 


396
ATGN{7}CGG
20
6
1
779


 


397
GGGN{20}GCA
59
30
1
780


 


398
CGGN{4}GGC
26
9
0.99
781


 


399
CGGN{16}AGC
22
7
0.99
782


 


400
CGGN{5}GGC
25
8
0.99
783


 


401
GCGN{0}GGA
25
8
0.98
784


 


402
GGCN{20}CAC
52
25
0.98
785


 


403
CCCN{9}CCC
97
58
0.97
786


 


404
ACCN{17}GGC
44
20
0.97
787


 


405
CCCN{6}CGA
18
5
0.96
788


 


406
AAGN{10}CGG
26
9
0.96
789


 


407
CGCN{17}CAC
21
6
0.95
790


 


408
CCCN{16}CGG
25
8
0.94
791


 


409
GACN{18}GGC
39
17
0.94
792


 


410
GGGN{15}GAC
47
22
0.92
793


 


411
GCCN{4}TCC
66
35
0.92
794


 


412
GGCN{15}CCC
56
28
0.92
795


 


413
CAGN{12}CGC
24
8
0.92
796


 


414
CCAN{3}GCG
22
7
0.91
797


 


415
CCGN{16}GAG
22
7
0.9
798


 


416
AGCN{2}CGC
24
8
0.89
799


 


417
GAGN{4}CCC
54
27
0.89
800


 


418
AGGN{3}CGC
23
7
0.88
801


 


419
CACN{13}AGG *
67
36
0.88
802


 


420
CCCN{4}CAG
88
51
0.88
803


 


421
CCCN{2}GAA
63
33
0.87
804


 


422
CGCN{19}GAG
21
6
0.87
805


 


423
ACGN{18}GGG
21
6
0.87
806


 


424
CCCN{4}GGC
62
32
0.87
807


 


425
CGGN{9}GAG
28
10
0.86
808


 


426
CCCN{3}GGG
66
26
0.86
809


 


427
GAGN{4}GGC
66
35
0.85
810


 


428
CGCN{5}GAG
18
5
0.84
811


 


429
CCGN{20}AGG
24
8
0.84
812


 


430
CCCN{15}CCC
88
51
0.83
813


 


431
AGGN{17}CCG
25
8
0.82
814


 


432
AGGN{6}GGG
89
52
0.82
815


 


433
GGCN{20}CCC
57
29
0.82
816


 


434
GCAN{17}CGC
19
5
0.82
817


 


435
CGAN{11}ACG
9
1
0.81
818


 


436
CGCN{2}GGA
19
5
0.81
819


 


437
CTGN{5}CCC
79
45
0.8
820


 


438
TCCN{20}CCA
77
43
0.8
821


 


439
CCAN{2}GGG
59
30
0.8
822


 


440
CCGN{15}GCG
14
3
0.8
823


 


441
CCAN{5}GGG
69
38
0.79
824


 


442
CGGN{1}TGC
24
8
0.79
825


 


443
CCCN{14}GCG
21
6
0.79
826


 


444
CAGN{0}CCG
27
10
0.79
827


 


445
GCCN{9}TCC
60
31
0.78
828


 


446
AGGN{20}CGC
22
7
0.78
829


 


447
CCCN{6}GAC
42
19
0.77
830


 


448
CGGN{11}CCA
23
7
0.76
831


 


449
GGGN{14}CAC
57
29
0.75
832


 


450
GCAN{15}CGC
19
5
0.74
833


 


451
CGCN{2}ACA
20
6
0.74
834


 


452
ACCN{9}CCC
57
29
0.73
835


 


453
GCGN{9}CGC
20
3
0.73
836


 


454
CAGN{15}GCG
23
7
0.73
837


 


455
CCCN{18}GTC
45
21
0.72
838


 


456
GCGN{3}CCC
24
8
0.72
839


 


457
CGGN{11}GCC
23
8
0.72
840


 


458
CCCN{1}CGG
24
8
0.71
841


 


459
GCCN{4}CCA
70
38
0.71
842


 


460
CCCN{4}CCG
30
12
0.7
843


 


461
CGTN{2}GCA
21
6
0.7
844


 


462
AGCN{7}TCG
18
5
0.69
845


 


463
CCGN{15}GAA
20
6
0.69
846


 


464
ACCN{5}CCC
62
33
0.69
847


 


465
CGCN{14}GAG
19
5
0.68
848


 


466
CCCN{7}CGC
30
12
0.68
849


 


467
GAGN{12}CGC
21
6
0.68
850


 


468
GGCN{17}CCC
58
30
0.67
851


 


469
ACGN{11}CTC
21
7
0.65
852


 


470
ACAN{9}CGG
24
8
0.65
853


 


471
CTGN{7}CCC
82
47
0.65
854


 


472
CCCN{2}GCC
72
40
0.65
855


 


473
CGGN{2}GCA
24
8
0.64
856


 


474
CCCN{0}TGC
83
48
0.64
857


 


475
CGCN{7}ACC
18
5
0.63
858


 


476
GCAN{2}GCC
54
27
0.63
859


 


477
GCGN{8}CCA
20
6
0.63
860


 


478
AGCN{0}CGC
22
7
0.63
861


 


479
GCGN{2}GCA
18
5
0.63
862


 


480
CCGN{2}GTC
18
5
0.62
863


 


481
CCGN{3}ACA
21
7
0.62
864


 


482
ACGN{13}TGG
21
7
0.62
865


 


483
CCAN{8}CGC
23
8
0.62
866


 


484
CCGN{9}GGC
23
8
0.61
867


 


485
CCAN{5}CCG
25
9
0.61
868


 


486
AGGN{3}GGG
97
59
0.61
869


 


487
CAGN{2}GGC
78
45
0.61
870


 


488
CCCN{8}CAG
81
47
0.61
871


 


489
AGCN{5}CAG
80
46
0.6
872


 


490
CGGN{16}GCC
22
7
0.6
873


 


491
GCGN{15}CCC
23
8
0.6
874


 


492
CCCN{11}GCC
59
31
0.59
875


 


493
CGAN{2}ACG
9
1
0.59
876


 


494
CGGN{4}GCC
22
7
0.59
877


 


495
CACN{6}CGC
19
6
0.59
878


 


496
CGGN{5}ACG
11
2
0.59
879


 


497
CTGN{4}GCC *
66
36
0.59
880


 


498
GGGN{18}CGA
18
5
0.59
881


 


499
CCTN{8}CGC
22
7
0.59
882


 


500
GCCN{4}CCC
67
37
0.58
883


 


501
CGGN{10}GCC
22
7
0.58
884


 


502
GCCN{5}GGA
54
27
0.57
885


 


503
ACCN{7}GCG
15
4
0.57
886


 


504
CCCN{8}CGC
24
8
0.57
887


 


505
CAGN{5}CCC
77
44
0.56
888


 


506
CACN{14}GGA
63
34
0.56
889


 


507
CCCN{1}GCC
94
57
0.55
890


 


508
CCCN{5}AGC
67
37
0.55
891


 


509
GGCN{5}GGA
59
31
0.55
892


 


510
CGAN{17}GAG
19
6
0.55
893


 


511
CGCN{7}ACA
18
5
0.54
894


 


512
CCAN{13}CCC
87
52
0.54
895


 


513
CGGN{20}GGC
24
8
0.54
896


 


514
CCCN{17}GCC
58
30
0.53
897


 


515
CCTN{10}CCG
30
12
0.53
898


 


516
CCCN{8}CCG
27
10
0.53
899


 


517
CGCN{3}GAG
18
5
0.52
900


 


518
CGCN{7}AAG
17
5
0.51
901


 


519
CGGN{11}GGA
23
8
0.51
902


 


520
CCGN{15}CCG
15
4
0.51
903


 


521
CCCN{3}GCA
57
30
0.51
904


 


522
CGGN{2}CAG
24
8
0.5
905


 


523
AGGN{2}CCG
24
8
0.5
906


 


524
CCCN{4}CAC
69
38
0.5
907


 


525
GGAN{19}CCC
56
29
0.49
908


 


526
CCCN{8}CAC
68
38
0.49
909


 


527
ACCN{6}CCG
18
5
0.49
910


 


528
CCCN{6}GGC
54
28
0.49
911


 


529
CCCN{6}CCG
29
11
0.48
912


 


530
CGCN{14}GCC
26
9
0.47
913


 


531
CCGN{5}TCC
25
9
0.46
914


 


532
GCCN{6}GCC
55
28
0.46
915


 


533
CGGN{7}GGA
24
8
0.45
916


 


534
GGGN{6}GGA
87
52
0.44
917


 


535
GCCN{12}TCC
60
32
0.44
918


 


536
AGTN{16}CCG
17
5
0.44
919


 


537
GGCN{19}GCC
68
29
0.44
920


 


538
CCGN{3}CCG
22
7
0.44
921


 


539
CCCN{8}ACC
58
31
0.44
922


 


540
CAGN{15}GCC
77
44
0.44
923


 


541
CCCN{17}CGG
24
8
0.44
924


 


542
GCGN{1}CCA
22
7
0.44
925


 


543
CCCN{14}CAG
79
46
0.44
926


 


544
CCCN{8}CCC
89
53
0.44
927


 


545
ACAN{12}GCG
23
8
0.43
928


 


546
AGGN{4}CCG
23
8
0.43
929


 


547
CGCN{13}GCC
23
8
0.43
930


 


548
GAGN{2}CGC
23
8
0.42
931


 


549
CCCN{9}GCG
21
7
0.42
932


 


550
CGCN{17}ACA
17
5
0.42
933


 


551
GCGN{17}CCA
23
8
0.42
934


 


552
AAGN{18}CCG
20
6
0.42
935


 


553
CGCN{1}GGA
18
5
0.41
936


 


554
CCAN{1}CCC
90
54
0.41
937


 


555
CGTN{18}TGC
20
6
0.41
938


 


556
TCCN{14}CGA
17
5
0.41
939


 


557
CACN{5}GGG
56
29
0.4
940


 


558
CCGN{12}GCA
21
7
0.4
941


 


559
CTGN{6}CCC
77
44
0.4
942


 


560
CGGN{8}GGC
32
13
0.4
943


 


561
CCAN{11}GGG
68
38
0.4
944


 


562
ACGN{19}CAA
21
7
0.39
945


 


563
GGGN{20}CCC
72
31
0.39
946


 


564
CGCN{3}CAG
23
8
0.39
947


 


565
AGCN{17}GGG
58
31
0.37
948


 


566
CACN{20}CCG
21
7
0.37
949


 


567
ACGN{17}CAG
24
8
0.37
950


 


568
AGGN{1}CCC
60
32
0.37
951


 


569
CGTN{12}CAC
20
6
0.37
952


 


570
CGGN{9}GGC
23
8
0.37
953


 


571
CGCN{10}GCG
18
3
0.37
954


 


572
CCCN{6}CTC
80
47
0.36
955


 


573
CCGN{10}AGG
23
8
0.36
956


 


574
CCCN{18}CAG
79
46
0.36
957


 


575
AGCN{17}CCG
21
7
0.36
958


 


576
AGCN{9}GCG
18
5
0.36
959


 


577
CCAN{3}GGC
62
34
0.36
960


 


578
CCCN{11}GGC
57
30
0.35
961


 


579
ACGN{5}GCA
23
8
0.35
962


 


580
CCCN{14}CGG
23
8
0.35
963


 


581
CCCN{5}CCA
91
55
0.35
964


 


582
CCGN{1}AGG
22
7
0.34
965


 


583
GGGN{10}GAC
45
22
0.34
966


 


584
CGCN{15}CCA
20
6
0.34
967


 


585
CCTN{19}CGC
22
7
0.34
968


 


586
CGTN{3}CGC
10
2
0.33
969


 


587
AGCN{14}CCG
21
7
0.33
970


 


588
GGCN{2}CGA
17
5
0.33
971


 


589
CAGN{8}CCC
79
46
0.33
972


 


590
CCGN{2}GAC
16
4
0.33
973


 


591
AGCN{19}AGG
70
40
0.32
974


 


592
CCTN{4}GGC
64
35
0.32
975


 


593
CCGN{11}AGC
22
7
0.32
976


 


594
CACN{4}CGC
18
5
0.32
977


 


595
CCGN{1}CCC
30
12
0.31
978


 


596
CTGN{13}GGC
73
42
0.31
979


 


597
CGCN{16}ACC
15
4
0.31
980


 


598
CACN{18}CAG
79
46
0.31
981


 


599
GGCN{8}GCC
68
29
0.29
982


 


600
GGGN{15}GGA
78
46
0.29
983


 


601
CCGN{16}GCC
22
7
0.29
984


 


602
CCGN{20}ACC
18
5
0.29
985


 


603
CGAN{7}CCC
17
5
0.28
986


 


604
CCGN{6}CTC
23
8
0.28
987


 


605
CGGN{10}CTC
22
7
0.28
988


 


606
CAGN{16}CGC
23
8
0.28
989


 


607
CCAN{3}AGG
77
45
0.27
990


 


608
GCCN{18}GCC
52
27
0.27
991


 


609
CGCN{18}GGA
19
6
0.26
992


 


610
CCGN{20}GGC
22
7
0.26
993


 


611
ACAN{10}GCG
17
5
0.26
994


 


612
CGGN{5}CCC
25
9
0.25
995


 


613
CCCN{7}TCC
75
43
0.25
996


 


614
ACGN{10}CGC
10
2
0.25
997


 


615
CCCN{3}TCC
81
48
0.25
998


 


616
CCGN{8}CGG
20
3
0.24
999


 


617
CCAN{15}CGG
22
7
0.24
1000


 


618
CCGN{6}CCG
17
5
0.24
1001


 


619
CAGN{9}GCG
25
9
0.24
1002


 


620
GAGN{1}CCC
62
34
0.24
1003


 


621
CCGN{18}TGC
22
7
0.23
1004


 


622
CCCN{7}CCA
85
51
0.23
1005


 


623
CGGN{3}CCA
24
9
0.23
1006


 


624
ACGN{1}CCC
18
5
0.23
1007


 


625
CGGN{13}TGA
21
7
0.22
1008


 


626
CTCN{6}GGC
53
28
0.22
1009


 


627
GCGN{2}GAC
15
4
0.22
1010


 


628
GGGN{11}ACC
49
25
0.22
1011


 


629
CGCN{4}GGA
17
5
0.22
1012


 


630
CCCN{11}CCG
27
10
0.22
1013


 


631
CCGN{19}GCA
20
6
0.22
1014


 


632
GCGN{0}GCA
20
6
0.21
1015


 


633
AGAN{7}CCC
61
33
0.21
1016


 


634
CGGN{2}CCA
21
7
0.21
1017


 


635
CCCN{7}CCC
89
54
0.21
1018


 


636
ACCN{4}GCG
15
4
0.2
1019


 


637
CCTN{15}CGC
20
6
0.2
1020


 


638
AGCN{9}GTC
44
21
0.2
1021


 


639
CCCN{18}CTC
74
43
0.2
1022


 


640
CGCN{18}CGA
9
1
0.19
1023


 


641
CCCN{15}GCC
62
34
0.18
1024


 


642
ACCN{11}GGC
45
22
0.18
1025


 


643
AGGN{15}CGC
29
12
0.18
1026


 


644
GCGN{0}CCA
27
10
0.18
1027


 


645
GCGN{9}AGC
18
5
0.17
1028


 


646
GGGN{18}GCA
59
32
0.17
1029


 


647
CCCN{17}CAG
77
45
0.17
1030


 


648
CCAN{8}CGG
22
8
0.16
1031


 


649
CCGN{10}GGC
21
7
0.16
1032


 


650
GCAN{0}GCC
76
44
0.16
1033


 


651
CAGN{2}CGC
20
6
0.16
1034


 


652
CGCN{8}GGC
19
6
0.16
1035


 


653
CTGN{17}GGC
65
36
0.16
1036


 


654
GGGN{14}ACC
46
23
0.16
1037


 


655
CCGN{1}TGC
20
6
0.16
1038


 


656
CAGN{8}CGC
22
8
0.15
1039


 


657
AAGN{11}CGC
17
5
0.15
1040


 


658
CCGN{6}TCC
22
8
0.14
1041


 


659
CCAN{18}CCC
72
42
0.14
1042


 


660
CCAN{0}CCC
84
51
0.14
1043


 


661
GAGN{6}CCC
53
28
0.14
1044


 


662
AGCN{20}GGC
52
27
0.14
1045


 


663
CAGN{0}CGC
21
7
0.14
1046


 


664
CCGN{12}CTC
22
8
0.14
1047


 


665
CGCN{15}ACG
9
1
0.13
1048


 


666
GGCN{17}CGA
15
4
0.13
1049


 


667
CCGN{16}AAG
19
6
0.13
1050


 


668
CGCN{14}TCC
19
6
0.12
1051


 


669
AGGN{7}CGC
20
7
0.12
1052


 


670
CGGN{7}CCC
22
8
0.12
1053


 


671
CGCN{4}GCC
34
15
0.12
1054


 


672
CGAN{6}CCC
17
5
0.12
1055


 


673
CCCN{19}GGA
60
33
0.11
1056


 


674
CCCN{16}GCG
28
11
0.11
1057


 


675
CCAN{7}CGC
20
7
0.11
1058


 


676
CCCN{6}GCC
80
48
0.11
1059


 


677
GCCN{14}TCC
55
29
0.11
1060


 


678
AGGN{14}GCC
64
36
0.1
1061


 


679
CGCN{11}GCC
20
7
0.1
1062


 


680
TCCN{0}GCA
17
5
0.09
1063


 


681
GCGN{8}CCC
27
11
0.09
1064


 


682
CCAN{11}GCG
19
6
0.09
1065


 


683
CACN{4}GGG
51
26
0.09
1066


 


684
CGGN{7}TCC
20
7
0.09
1067


 


685
GCGN{5}GCC
20
7
0.09
1068


 


686
ACGN{12}CAG
26
10
0.09
1069


 


687
CCGN{19}CGC
14
4
0.08
1070


 


688
CGGN{8}TGC
18
5
0.08
1071


 


689
CCCN{1}GAG
65
37
0.07
1072


 


690
GCGN{19}TGA
18
6
0.07
1073


 


691
GGCN{15}GCC
70
31
0.07
1074


 


692
CCGN{7}CCC
27
11
0.07
1075


 


693
ACAN{19}CCC
63
35
0.07
1076


 


694
ACCN{16}GGG
47
24
0.07
1077


 


695
AGAN{1}GGC
64
36
0.07
1078


 


696
GGGN{17}TGA
64
36
0.06
1079


 


697
CAGN{5}GGG
83
50
0.06
1080


 


698
GCCN{13}CGC
22
8
0.06
1081


 


699
GCGN{7}GGA
19
6
0.06
1082


 


700
CAGN{14}CCA
94
58
0.06
1083


 


701
CCGN{4}GTC
16
4
0.06
1084


 


702
CCCN{13}CGC
22
8
0.06
1085


 


703
GCGN{14}ACC
15
4
0.05
1086


 


704
CAGN{20}GGG
81
49
0.05
1087


 


705
CCGN{4}CCC
27
11
0.05
1088


 


706
CGCN{5}GGC
18
6
0.05
1089


 


707
CCTN{6}GGC
57
31
0.05
1090


 


708
AGGN{3}GGC
67
38
0.05
1091


 


709
CGGN{11}CGC
14
4
0.05
1092


 


710
CTGN{18}GGA
77
46
0.04
1093


 


711
CACN{17}CCA
74
43
0.04
1094


 


712
CGGN{3}GAG
22
8
0.04
1095


 


713
CCCN{9}CCA
82
49
0.03
1096


 


714
CCCN{1}ACG
18
6
0.03
1097


 


715
CAGN{1}GCC
72
42
0.03
1098


 


716
AGGN{6}CCG
23
8
0.03
1099


 


717
AGCN{9}GGG
57
31
0.03
1100


 


718
CCCN {7}GGG
54
29
0.02
1101


 


719
CCTN{13}CCC
88
54
0.02
1102


 


720
CCGN{19}TTC
20
7
0.02
1103


 


721
CCCN{7}CCG
27
11
0.02
1104


 


722
CGAN{6}GGC
17
5
0.01
1105


 


723
CGGN{4}CTC
21
7
0.01
1106


 


724
CGGN{0}CGC
13
3
0.01
1107


 


725
CCTN{13}ACG
19
6
0.01
1108


 


726
GGGN{6}CAC
53
28
0.01
1109


 


727
CCCN{16}CGC
21
7
0.01
1110


 


728
CCCN{10}CTC
76
45
0
1111


 


729
CCCN{0}CAG
92
57
0
1112


 


730
GCCN{5}CCC
65
37
0
1113





The patterns are ranked according to significance coefficient. These were determined using RSA-Tools with the random sequence from the human genome as reference. Patterns that comprise the most discriminant variables in Linear Discriminant Analysis are indicated with an asterisk (SEQ ID NOS:384-1113).













TABLE 6







STAR elements, including genomic location


and length (SEQ ID NOS: 1-66)










STAR
Location1
Length2
SEQ ID NO:













 1
2q31.1
750
1


 2
7p15.2
916
2


  33
15q11.2 and 10q22.2
2132
3


 4
1p31.1 and 14q24.1
1625
4


  54
20q13.32
1571
5


 6
2p21
1173
6


 7
1q34
2101
7


 8
9q32
1839
8


  94
10p15.3
1936
9


10
Xp11.3
1167
10


11
2p25.1
1377
11


12
5q35.3
1051
12


 134
9q34.3
1291
13


 144
22q11.22
732
14


15
1p36.31
1881
15


16
1p21.2
1282
16


17
2q31.1
793
17


18
2q31.3
497
18


19
6p22.1
1840
19


20
8p13.3
780
20


21
6q24.2
620
21


22
2q12.2
1380
22


23
6p22.1
1246
23


24
1q21.2
948
24


 255
1q21.3
1067
25


26
1q21.1
540
26


27
1q23.1
1520
27


28
22q11.23
961
28


29
2q13.31
2253
29


30
22q12.3
1851
30


31
9q34.11 and 22q11.21
1165
31


32
21q22.2
771
32


33
21q22.2
1368
33


34
9q34.14
755
34


35
7q22.3
1211
35


36
21q22.2
1712
36


37
22q11.23
1331
37


38
22q11.1 and 22q11.1
~1000
38


39
22q12.3
2331
39


40
22q11.21
1071
40


41
22q11.21
1144
41


42
22q11.1
735
42


43
14q24.3
1231
43


44
22q11.1
1591
44


45
22q11.21
1991
45


46
22q11.23
1871
46


47
22q11.21
1082
47


48
22q1.22
1242
48


49
Chr 12 random clone, and 3q26.32
1015
49


50
6p21.31
2361
50


51
5q21.3
2289
51


52
7p15.2
1200
52


53
Xp11.3
1431
53


54
4q21.1
981
54


55
15q13.1
501
55


56
includes 3p25.3
741
56


57
4q35.2
1371
57


58
21q11.2
1401
58


59
17 random clone
872
59


60
4p16.1 and 6q27
2068
60


61
7p14.3 and 11q25
1482
61


62
14q24.3
1011
62


63
22q13.3
1421
63


64
17q11.2
1414
64


65
7q21.11 = 28.4
1310
65


66
20q13.33 and 6q14.1
~2800
66





1Chromosomal location is determined by BLAST search of DNA sequence data from the STAR elements against the human genome database. The location is given according to standard nomenclature referring to the cytogenetic ideogram of each chromosome; e.g., 1p2.3 is the third cytogenetic sub-band of the second cytogenetic band of the short arm of chromosome 1 (WorldWideWeb.ncbi.nlm.nih.gov/Class/MLACourse/Genetics/chrombanding.html). In cases where the forward and reverse sequencing reaction identified DNAs from different genomic loci, both loci are shown.


2Precise lengths are determined by DNA sequence analysis; approximate lengths are determined by restriction mapping.


3Sequence and location of STAR3 (SEQ ID NO: 3) has been refined since assembly of Tables 2 and 4 of EP 01202581.3.


4The STARs with these numbers in Tables 2 and 4 of EP 01202581.3 have been set aside (hereafter referred to as “oldSTAR5” etc.) and their numbers assigned to the STAR elements shown in the DNA sequence appendix. In the case of oldSTAR5, oldSTAR14, and oldSTAR16, the cloned DNAs were chimeras from more than two chromosomal locations; in the case of oldSTAR9 and oldSTAR13, the cloned DNAs were identical to STAR4 (SEQ ID NO: 4).


5Identical to Table 4 “STAR18” (SEQ ID NO: 18) of EP 01202581.3.













TABLE 7







STAR elements convey stability over


time on transgene expression1










Cell Divisions2
Luciferase Expression3













STAR6 (SEQ ID NO: 6)
42
18,000


plus puromycin
60
23,000



84
20,000



108
16,000


STAR6 (SEQ ID NO: 6)
84
12,000


without puromycin4
108
15,000



144
12,000





1Plasmid pSDH-Tet-STAR6 was transfected into U-2 OS cells, and clones were isolated and cultivated in doxycycline-free medium. Cells were transferred to fresh culture vessels weekly at a dilution of 1:20.


2The number of cell divisions is based on the estimation that in one week the culture reaches cell confluence, which represents ~6 cell divisions.


3Luciferase was assayed as described in Example 4.


4After 60 cell divisions the cells were transferred to two culture vessels; one was supplied with culture medium that contained puromycin, as for the first 60 cell divisions, and the second was supplied with culture medium lacking antibiotic.













TABLE 8







Human STAR elements and their putative


mouse orthologs and paralogs












Number
STAR
Human1
Mouse2
Similarity3
SEQ ID NO:
















1
  1
2q31.1
2D
600
bp 69%
1


2
 2
7p15.2
6B3
909
bp 89%
2


3
3a
5q33.3
11B2
248
bp 83%
3


4
3b
10q22.2
14B
1. 363
bp 89%
3






2. 163
bp 86%


5
 6
2p21
17E4
437
bp 78%
6


6
12
5q35.3
11b1.3
796
bp 66%
12


7
13
9q34.3
2A3
753
bp 77%
13


8
18
2q31.3
2E1
497
bp 72%
18


9
36
21q22.2
16C4
166
bp 79%
36


10
40
22q11.1
6F1
1. 270
bp 75%
40






2. 309
bp 70%


11
50
6p21.31
17B1
1. 451
bp 72%
50






2. 188
bp 80%






3. 142
bp 64%


12
52
7p15.2
6B3
1. 846
bp 74%
52






2. 195
bp 71%


13
53
Xp11.3
XA2
364
bp 64%
53


14
54
4q21.1
5E3
1. 174
bp 80%
54






2. 240
bp 73%






3. 141
bp 67%






4. 144
bp 68%


15
 61a
7p14.3
6B3
188
bp 68%
61





1Cytogenetic location of STAR element in the human genome.


2Cytogenetic location of STAR element ortholog in the mouse genome.


3Length of region(s) displaying high sequence similarity, and percentage similarity. In some cases more than one block of high similarity occurs; in those cases, each block is described separately. Similarity <60% is not considered significant.













TABLE 9







Candidate STAR elements tested by Linear


Discriminant Analysis (SEQ ID NOS: 66-84)












Candidate






STAR
Location1
Length
SEQ ID NO:







T2 F
20q13.33
~2800
66



T2 R
6q14.1
~2800
67



T3 F
15q12
~2900
68



T3 R
7q31.2
~2900
69



T5 F
9q34.13
 ND2
70



T5 R
9q34.13
ND
71



T7
22q12.3
~1200
72



T9 F
21q22.2
~1600
73



T9 R
22q11.22
~1600
74



T10 F
7q22.2
~1300
75



T10 R
6q14.1
~1300
76



T11 F
17q23.3
~2000
77



T11 R
16q23.1
~2000
78



T12
4p15.1
~2100
79



T13 F
20p13
~1700
80



T13 R
1p13.3
~1700
81



T14 R
11q25
~1500
82



T17
2q31.3
ND
83



T18
2q31.1
ND
84







1Chromosomal location is determined by BLAT search of DNA sequence data from the STAR elements against the human genome database. The location is given according to standard nomenclature referring to the cytogenetic ideogram of each chromosome; e.g., 1p2.3 is the third cytogenetic sub-band of the second cytogenetic band of the short arm of chromosome 1 (WorldWideWeb.ncbi.nlm.nih.gov/Class/MLACourse/Genetics/chrombanding.html). F, forward sequencing reaction result; R, reverse sequencing reaction result. When the forward and reverse sequencing results mapped to different genomic locations, each sequence was extended to the full length of the original clone (as determined by restriction mapping) based on sequence information from the human genome database.



2ND: Not Determined.













TABLE 10







Arabidopsis STAR elements of the invention, including


chromosome location and length (SEQ ID NOS: 85-119).












STAR
Chromosome
Length, kb
SEQ ID NO:
















A1
I
1.2
85



A2
I
0.9
86



A3
I
0.9
87



A4
I
0.8
88



A5
I
1.3
89



A6
I
1.4
90



A7
II
1.2
91



A8
II
0.8
92



A9
II
0.9
93



A10
II
1.7
94



A11
II
1.9
95



A12
II
1.4
96



A13
II
1.2
97



A14
II
2.1
98



A15
II
1.4
99



A16
II
0.7
100



A17
II
1.5
101



A18
III
1.5
102



A19
III
0.7
103



A20
III
2.0
104



A21
IV
1.8
105



A22
IV
0.8
106



A23
IV
0.6
107



A24
IV
0.5
108



A25
V
0.9
109



A26
V
1.9
110



A27
V
1.1
111



A28
V
1.6
112



A29
V
0.9
113



A30
V
2.0
114



A31
V
2.0
115



A32
V
1.3
116



A33
V
0.9
117



A34
I
0.9
118



A35
II
1.1
119










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## Claims

1. A cell comprising a first polypeptide expression unit and a second polypeptide expression unit, said first and said second polypeptide expression units each encoding at least one polypeptide of interest, wherein said first and second polypeptide expression units each comprise at least one sequence having the capacity to at least in part block chromatin-associated repression, wherein the sequence having the capacity to at least in part block chromatin-associated repression for the first polypeptide expression unit comprises SEQ ID NO: 17, and wherein the sequence having the capacity to at least in part block chromatin-associated repression for the second polypeptide expression unit is selected from the group consisting of any one of SEQ ID NO: 1 through SEQ ID NO: 65.

2. The cell of claim 1, wherein the sequence having the capacity to at least in part block chromatin-associated repression for the second polypeptide expression unit comprises SEQ ID NO: 17.

3. The cell of claim 1, wherein said first and second polypeptide expression units each further encode a different selection marker.

4. The cell of claim 1, wherein at least one of said first and second polypeptide expression units comprises a monocistronic gene comprising an open reading frame encoding a polypeptide of interest and wherein said monocistronic gene is under control of a functional promoter.

5. The cell of claim 1, wherein at least one of said first and second polypeptide expression units comprises a bicistronic gene comprising in the following order: (i) an open reading frame encoding a polypeptide of interest, (ii) an Internal Ribosome Entry Site (IRES), and (iii) a selection marker, and wherein said bicistronic gene is under control of a functional promoter.

6. The cell of claim 1, wherein at least one of said first and second polypeptide expression units comprises at least two of the sequences having the capacity to at least in part block chromatin-associated repression, arranged such that said polypeptide expression unit is flanked on both sides by at least one of the sequences having the capacity to at least in part block chromatin-associated repression.

7. The cell of claim 1, wherein at least one polypeptide of interest comprises an immunoglobulin heavy chain and the other polypeptide of interest comprises an immunoglobulin light chain, wherein said heavy and light chain can form a functional antibody.

8. A method for expressing at least two polypeptides of interest in a cell, said method comprising:
providing a cell, the cell comprising a first polypeptide expression unit and a second polypeptide expression unit, said first and second polypeptide expression units each encoding at least one polypeptide of interest, wherein said first and second polypeptide expression units each comprise at least one sequence having the capacity to at least in part block chromatin-associated repression, wherein the sequence having the capacity to at least in part block chromatin-associated repression for the first polypeptide expression unit comprises SEQ ID NO: 17, and wherein the sequence having the capacity to at least in part block chromatin-associated repression for the second polypeptide expression unit is selected from the group consisting of: any one of SEQ ID NO: 1 through SEQ ID NO: 65; and
culturing the cell under conditions wherein said first and second polypeptide expression units are expressed.

9. The method according to claim 8, wherein the sequence having the capacity to at least in part block chromatin-associated repression for the second polypeptide expression unit comprises SEQ ID NO: 17.

10. The method according to claim 8, wherein said first and second polypeptide expression units each further encode a different selection marker.

11. The method according to claim 8, wherein at least one of said first and second polypeptide expression units comprises a monocistronic gene comprising an open reading frame encoding a polypeptide of interest and wherein said monocistronic gene is under control of a functional promoter.

12. The method according to claim 8, wherein at least one of said first and second polypeptide expression units comprises a bicistronic gene comprising in the following order: (i) an open reading frame encoding a polypeptide of interest, (ii) an Internal Ribosome Entry Site (IRES), and (iii) a selection marker, and wherein said bicistronic gene is under control of a functional promoter.

13. The method according to claim 8, wherein at least one of said first and second polypeptide expression units comprises at least two of the sequences having the capacity to at least in part block chromatin-associated repression, arranged such that said polypeptide expression unit is flanked on both sides by at least one of the sequences having the capacity to at least in part block chromatin-associated repression.

14. The method according to claim 8, wherein at least one polypeptide of interest comprises an immunoglobulin heavy chain and the other polypeptide of interest comprises an immunoglobulin light chain, wherein said heavy and light chain can form a functional antibody.

15. A polypeptide expression unit comprising:
a bicistronic gene comprising in the following order: (i) an open reading frame encoding a polypeptide of interest, (ii) an Internal Ribosome Entry Site (IRES), and (iii) a selection marker, and wherein said bicistronic gene is under control of a functional promoter; and
at least one sequence having the capacity to at least in part block chromatin-associated repression, wherein the sequence having the capacity to at least in part block chromatin-associated repression comprises SEQ ID NO: 17.

16. The polypeptide expression unit of claim 15, further comprising a further sequence having the capacity to at least in part block chromatin-associated repression, wherein said further sequence is selected from the group consisting of any one of SEQ ID NO: 1 through SEQ ID NO: 65, and wherein said further sequence is arranged with said polypeptide expression unit such that said polypeptide expression unit, on one side, comprises SEQ ID NO: 17, and on another side comprises said further sequence having the capacity to at least in part block chromatin-associated repression.

17. A method for expressing two polypeptides of interest, the method comprising:
a) providing host cells comprising:
(i) a first polypeptide expression unit comprising a bicistronic gene comprising a promoter functionally linked to a sequence encoding a first polypeptide of interest and a first selectable marker gene, and
(ii) a second polypeptide expression unit comprising a bicistronic gene comprising a promoter functionally linked to a sequence encoding a second polypeptide of interest and a second selectable marker gene,

wherein said second selectable marker gene is different from said first selectable marker gene, and wherein said first polypeptide expression unit, or said second polypeptide expression unit, or each of said first and said second polypeptide expression units comprise at least one sequence having the capacity to at least in part block chromatin-associated repression, wherein the sequence having the capacity to at least in part block chromatin-associated repression comprises SEQ ID NO: 17;
b) selecting a host cell by selecting for expression of said first and second selectable marker genes; and
c) culturing a selected host cell to express said first and second polypeptides.

18. The method according to claim 17, wherein each of said first and said second expression units comprise at least one sequence having the capacity to at least in part block chromatin-associated repression, the sequence having the capacity to at least in part block chromatin-associated repression comprising SEQ ID NO: 17.

19. The method according to claim 17, wherein the two polypeptides of interest form part of a multimeric protein.

20. A set of two polypeptide expression units, said set comprising:
(i) a first polypeptide expression unit comprising a bicistronic gene comprising a promoter functionally linked to a sequence encoding a first polypeptide of interest and a first selectable marker gene, and
(ii) a second polypeptide expression unit comprising a bicistronic gene comprising a promoter functionally linked to a sequence encoding a second polypeptide of interest and a second selectable marker gene,
wherein said second selectable marker gene is different from said first selectable marker gene, and wherein said first polypeptide expression unit, or said second polypeptide expression unit, or both said first and said second polypeptide expression units comprise at least one sequence having the capacity to at least in part block chromatin-associated repression, wherein the sequence having the capacity to at least in part block chromatin-associated repression comprises SEQ ID NO: 17.

21. The set of two polypeptide expression units of claim 20, wherein both said first and second polypeptide expression unit have integrated into the genome of a cell.