INTEGRATION CASSETTE FOR IMPROVEMENT OF TRANSGENESIS IN EUKARYOTES

Abstract: The present invention relates to DNA constructs provided to improve transgenesis or genomic integration of DNA fragment of interest into nuclear DNA of eukaryotic cell. The DNA constructs of the present invention allow a more efficient procedure by increasing the rate of genomic integration of a DNA fragment. After integration into host cell's DNA, the DNA constructs may provide a mean of screening transgenic cells. An increasing integration means, as well as a method for screening transgenic cells with said DNA construct are disclosed.

Description: INTEGRATION CASSETTE FOR IMPROVEMENT OF TRANSGENESIS IN EUKARYOTES BACKGROUND OF THE INVENTION (a) Field of the Invention The invention relates to a method of transform¬ ing eukaryotic cells. More particularly, this inven¬ tion relates to the use of DNA constructs designed to insert a particular DNA fragment more efficiently into host cell's DNA in the goal of transgenesis. (b) Description of Prior Art Using recombinant DNA technology, foreign DNA sequences can be inserted into an organism's genome to alter the phenotype of the host's organism. A variety of different procedures have been described and are utilized to produce stably transformed eukaryotic cells. All of these procedures are based on, first introducing the foreign DNA into the eukaryotic cell, and followed by isolation of those cells containing the foreign DNA into the eukaryotic cell's DNA. Unfortunately, to produce transgenic animal and plant, all current higher eukaryotic cell transforma¬ tion procedures produce in very low proportions trans¬ formed germinal (oocytes, spermatozoa, zygotes, sperma- togonia, blastomers, etc.) or stem cells that contain the introduced foreign DNA inserted throughout the genome. Additionally, the random insertion of intro¬ duced DNA into the genome of host cells can be lethal if the foreign DNA happens to insert into, and thus mutate, a unique vital native gene in a critical man- ner. Introduction of foreign DNA sequence in the mouse and other laboratory animals is now relatively easy to perform and is currently used in transgenesis. At the moment, progress in adapting the technology to higher plants and animals, particularly to commercially exploited animals (e.g. farm animals), has not reached the integration rate of foreign DNA observed in mice. In mice, transgenesis occurs in about six percent of the zygotes injected, whereas it is only 0.6% in pigs, 0.7% in sheep and 0.5% in cattle. A technique currently exist for selecting cells after homologous recombination (HR) event between an endogenous gene and a DNA construct carrying a copy of the gene (Smitties et al., 1985, Nature, 317:230). This work has been extended for the replacement of a targeted gene or to cause gene deletion as well as introduction of foreign DNA molecules (Thomas et al., 1987, Cell , 51:503). In general, one homologous recom¬ bination (HR) event occurs for every 10 2 to 10 5 non- homologous integration events. The HR approach was also tested for mouse transgenesis by microinjecting the DNA construct directly into the fertilized oocytes (Brinster et al . , 1989, Proc . Na tl . Acad. Sci . USA, 86:7087) The results demonstrated the feasibility of HR to correct a mutant gene which is inactive in fer- tilized mouse eggs. However, the gene deletion was corrected by HR in one out of five hundred (1/500) transgenic mice that incorporated the injected DNA. For all the above-mentioned reasons, it would be highly desirable to be provided with a transformation system which would allow integration of a DNA fragment by targeting a repeated site of the host's genome. Preferably, such a system would also provide a means of preventing any insertion into a vital gene or genetic region existing in a single copy. SUMMARY OF THE INVENTION One aim of the present invention is to improve the insertion of a length of DNA in the host germinal or embryonic stem cells. In addition, another aim of the present inven¬ tion is to provide for a proper selection of the tar¬ geted site can minimize position effects, enabling an inserted gene to synthesize an effective amount of its protein product. Therefore, the present invention allows a much more efficient system of improving the rate of trans¬ genesis in plants and animals than is currently possi¬ ble. in accordance with the present invention there is provided a DNA construct for inserting a DNA frag¬ ment of interest into eukaryotic host cell, the con¬ struct comprising an integration cassette flanked by site-specific recombination sequences in which is inserted the DNA of interest, wherein the DNA fragment of interest is flanked by a nucleotide sequence sharing homology to a nucleotide sequence present in more than one copy in the eukaryotic cell, whereby the integra¬ tion cassette improves the genomic insertion of the DNA fragment of interest in a site-specific manner. The DNA sequence of interest may be flanked by nucleotide sequences sharing homology to a repeated nucleotide sequence present in the eukaryotic cell, wherein the flanking nucleotide sequence being linked to only one extremity or to both extremities of a DNA fragment of interest to be integrated into a host cell's genome. Such flanking nucleotide sequences sharing homology with a DNA sequence of the host cell's genome may be selected from the group consisting of a Satellite DNA sequence, corticotropin-β-lipotropin, non-satellite repetitive DNA, histone, ribosomic RNA transfer RNA coding sequences, Pst, Bsu and Alu-like repetitive sequences. The DNA of interest may be linked to regulatory sequences capable of expressing the said DNA of inter¬ est in the eukaryotic cell. In accordance with the present invention there is also provided a method for improvement of the pro¬ duction of fertile, transgenic eukaryotic organisms wherein the transgenic eukaryotic organisms have a DNA sequence of interest integrated at a predetermined repeated DNA sequence of the organisms, the method com- prising the steps of introducing into eukaryotic cells a DNA construct comprising a gene of interest flanked by site-specific recombination sequences, wherein gene of interest is flanked by nucleotide sequences sharing homology to the predetermined nucleotide sequence pre- sent in the eukaryotic cell, and the gene of interest is operably linked to regulatory sequences capable of expressing the gene in the eukaryotic cell. In accordance with the present invention there is also provided a transgenic plant consisting essen- tially of a plant cell, seed or plant from the in vi tro introduction of an exogenous DNA fragment into a plant cell by the method of the present invention. In accordance with the present invention there is also provided a transgenic animal consisting essen- tially of an animal cell, gamete, zygote, blastomer, embryonic stem cell or animal from the in vi tro intro¬ duction of an exogenous DNA fragment into a plant cell by the method of the present invention. In accordance with the present invention there is also provided a method of directly selecting for insertion of a DNA sequence of interest into a specific sequence of an organism's DNA said method comprising the steps of a) introducing a DNA construct of the present invention into the cells of said organisms; b) choosing a pair of primers, wherein one primer recognizes a DNA sequences of the DNA fragment of interest, and the other primer recognizes a DNA sequence of the host cell, where the sequence recognized by the other primer exists outside of the DNA construct of claim 1; c) using the pair of primers of step b) to amplify the DNA construct inserted into the host cell's genome by homologous recombination; and d) selecting those cells having the DNA construct integrated into the DNA of the cell, thus being transgenic cells. Additional objects, features, and advantages of the invention will become apparent to those skilled in the art upon consideration of the following detailed description of preferred embodiments exemplifying the best mode of the invention as presently perceived. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1A-1C each illustrates a schematic repre¬ sentation of DNA constructs in accordance with the pre¬ sent invention, respectively with both extremities repeatedly, invertedly or partially homologous, 5' homologous and 3' homologous; Fig. 2 illustrates the synthesis of the con¬ structs in accordance with the present invention; and Fig. 3 illustrates the recognition sites of the oligonucleotide primers used in the PCR reactions in accordance with the present invention. DETAILED DESCRIPTION OF THE INVENTION In accordance with one preferred embodiment of the present invention, the transformation of eukaryotic cells is improved by the use of DNA sequences that are identical to a repeated predetermined sequence to the eukaryote' s DNA. Typically the introduced DNA sequence will constitute entire functional genes. Here eukaryotic cells includes all manipulated gametes, zygotes, embryos, blastomers or embryonic stem cells used to create a transgenic organism. Eukaryotic cells can also be transformed with other DNA sequences such as gene transcription and translation regulatory sequences. Transcription and translation regulatory sequences are those DNA sequences necessary for effi- cient expression of a gene product. In general such regulatory elements can be operably linked to any gene to control the gene's expression, the entire unit being referred to as the "expression cassette". An expres¬ sion cassette is intended to typically contain, in addition to the coding sequence, a promoter region, a translation initiation site and a translation termina¬ tion sequence. Selected endonuclease restriction sites may also be included at the ends of an expression cas¬ sette to allow the cassette to be easily inserted or removed when creating DNA constructs. The expression of a gene is primarily directed by its own promoter, although other DNA regulatory ele¬ ments are necessary for efficient expression of a gene product. Promoter sequence elements include the TATA box consensus sequence (TATAAT), which is usually 20 to 30 base pairs (bp) upstream of the transcription start site. In most instances the TATA box is required for accurate transcription initiation. Promoters can be either constitutive or induc- ible. A constitutive promoter controls transcription of a gene at a constant rate during the life of a cell, whereas an inducible promoter's activity fluctuates as determined by the presence (or the absence) of a spe¬ cific inducer. The regulatory elements of an inducible promoter are usually located further upstream of the transcriptional start site than the TATA box. Ideally, for experimental purposes in accordance with the pre¬ sent invention, an inducible promoter should possess each of the following properties: a low to non-existent basal level of expression in the absence of inducer, a high level of expression in the presence of inducer, and an induction scheme that does not otherwise alter the physiology of the cell. The basal transcriptional activity of all promoters can be increased by the pres- ence of an "enhancer" sequence. Although the mechanism is unclear, certain defined enhancer regulatory sequences are known, to those familiar with the art, to increase a promoter's transcription rate when the sequence is brought in proximity to the promoter. The creation of a transformed cell requires that the DNA be physically placed within the host cell. Current transformation procedures utilize a variety of techniques to introduce DNA into a cell. In one form of transformation, the DNA is microinjected directly into cells through the use of micropipettes. Alterna¬ tively, high velocity ballistics can be used to propel small DNA associated particles into the cell. In another form, the cell is permeabilized by the presence of polyethylene glycol, thus allowing DNA to enter the cell through diffusion. DNA can also be introduced into a cell by fusing protoplasts with there entities which contain DNA. These entities include minicells, cells, lysosomes or other fusible lipid-surfaced bodies. Transformed cells, those containing the DNA inserted into the host cell's DNA, can be selected from untransformed cells if a selectable marker is included as part of the introduced DNA sequences. Selectable markers include genes that provide antibiotic resis- tance (e.g. G418) or herbicide (e.g. kanamycin, hygro- mycin) resistance. Alternatively, conventional tech¬ niques like restriction enzyme digestion and Southern blot are generally used for the molecular analysis of events such as HR. However, in certain cases where genomic DNA of transformed cells is available in very low quantities, analysis by these conventional methods can make selection of transgenic cells unperformable. Amplification of targeted DNA sequence by polymerase chain reaction (PCR) allows detection in a sensitive and highly selective manner of this DNA fragment. PCR is an alternative method which has been used to reveal HR events in eukaryotic cells (Zimmer et al. , 1989, Na ture, 338:150). HR analysis by PCR which gives a desired response is an indication that introduced DNA construct is integrated in the targeted sequence of the host cell's DNA. All the transformation techniques described above have the limitation that they result in rare inserted copies, and result in foreign DNA inser¬ tions in very small number or cells, most particularly in gametes and zygotes. Therefore the proportions of transgenic organisms is low too. The present invention enables the increase of integration of a foreign DNA fragment in the host cell's genome, while it allows to the targeting of a length of DNA to a specific repeated site. Presumably the selected targeted site will also allow the inserted gene to produce its protein production an amount suffi¬ cient to produce the desired effect. In one preferred embodiment, the introduced DNA consists of combination in tandem arrangement of a pro¬ moter region linked to a gene or interest flanked by partial of total half-cut or repeated DNA fragments homologous to a sequences existing in the host cell's DNA. The length of the homologous fragments can vary from 2 to 10 thousands base pairs (bp). The first element of the invention involves the use of a DNA fragment identical to a specific repeated site in the host cell's genome to flank the DNA con¬ struct to introduce. Targeting can be carried out via homologous recombination. Homologous recombination is a reaction between any pair of DNA sequences having a similar sequence of nucleotides (homologous sequences, where the two sequences interact (recombine) to form a new recombinant DNA species. The frequency of homolo- gous recombination increases as the number of copies and the length of the shared nucleotide DNA sequence increases, and is higher with linearized plasmid mole¬ cules than with circularized plasmid molecules. Homologous recombination can occur between two DNA sequences that are less than identical, but the recom¬ bination frequency declines as the divergence between the two sequences increases. Introduced DNA sequences may be targeted via homologous recombination by linking the DNA of interest to sequences sharing homology with endogenous sequences of the host cell. Once the DNA enters the cell, the two homologous sequences can interact to insert the introduced DNA at the site where the homologous genomic DNA sequences were located. Therefore, the choice of homologous sequences that contain the introduced DNA will determine the rate and site at which the intro¬ duced DNA is integrated. For example, in the goal to produce transgenic animals, if the DNA of interest is linked to DNA sequences sharing homology to a single copy gene of the host eukaryotic cell, the DNA sequence of interest may be inserted via homologous recombina¬ tion in a very low proportion of manipulated cells. However, if the DNA sequence of interest is linked to DNA sequences sharing homology to a multi-copy DNA sequence of the host eukaryotic cell, then the DNA sequence of interest can be inserted in many specific sites where a copy of the genomic DNA sequence is located. Preferably, the predetermined host DNA site of insertion is a non-essential endogenous gene or other genomic DNA sequence present in high copy number, such as a DNA satellite sequence. Alternatively, the homologous region flanking the gene(s) of interest could exists in only two copies in the host cell's DNA. DNA can be inserted into the genome by a homolo- gous recombination reaction involving either a single reciprocal recombination (resulting in the insertion of the entire length of the introduced DNA) or through a double reciprocal recombination (resulting in the insertion of only the DNA located between the two recombination events). A single homologous recombina¬ tion event may then result in the entire introduced DNA sequence being inserted into the genomic gene. Alter¬ natively, a double recombination event can be achieved by flanking each end of the DNA sequence of interest (the sequence intended to be inserted into the genome) with DNA sequences homologous to the targeted genomic sequences. Although introduced sequences can be tar¬ geted for insertion a specific genomic site via homolo¬ gous recombination, in higher eukatyotes, homologous recombination is a relatively rare event compared to random insertion events. In plant cells, foreign DNA molecules find homologous sequences in the cell's genome and recombine at a frequency of 0.5 to 4.2 x 10 " 4 (the number of targeted events divided by the number of random integration events), while in animal one homologous recombination occurs against 10 2 to 10^ ran¬ dom recombinations. Thus the efficiency or production of transgenic plant or animal can be potentially increased by flanking a transgene with repeated host cell's DNA sequences. One way to control the activity of the transgene product is to link it to an inducible promoter. Induc¬ ible promoters include any promoter capable of increas¬ ing the amount of gene product produced, by a given gene, in response to exposure to an inducer. Inducible promoters are known to those familiar with the art and a variety exist that could conceivably be used to drive expression of the transgene. Two preferred inducible promoters are the heat shock promoter and the glucocorticoid system. Promot¬ ers regulated by heat shock, such as the promoter nor¬ mally associated with the gene encoding the 70-kDa heat shock protein, can increase expression several-fold after exposure to elevated temperatures. The heat shock promoter could be used as an environmentally inducible promoter for controlling transcription. The system consists of a gene encoding glucocorticoid receptor protein (GR) which in the presence of a ster¬ oid hormone forms a complex with the hormone. This complex then binds to a short nucleotide sequence (26bp) named the glucocorticoid response element (GRE), and this binding activates the expression of linked genes. As shown in Fig. 1, the transformation construct can also include a polylinker region located in the genomic DNA sequence. The addition of a polylinker region promotes the ease of constructing unique DNA molecules. Through the use of specific nucleotide restriction enzymes, gene cassettes and other DNA sequences can be inserted into the polylinker region. Any DNA sequence inserted into the polylinker can then be flanked with identical sequences of the host genome to improve insertion into a host cell's DNA via site specific recombination. Typically all the genes con- tained on the introduced DNA will be linked to a regu- latory sequences capable of expression the gene's prod¬ uct in a eukaryotic cell. As shown in Fig. 1, constructs can be physically linked to additional sequences to form a circularized DNA molecule (a plasmid). These additional sequences would contain a gene's cassette encoding a bacterial selectable marker and a bacterial origin of replica¬ tion. This plasmid is useful in generation large amounts of the DNA constructs. The procedure consists of using this plasmid to transform bacterial cells, growing the bacterial cells under selection for the presence of the plasmid and then finally isolating the plasmid from the replicated bacterial cells. In order to prevent integration of unnecessary plasmid DNA that is outside the site-specific recombination sequences, the plasmid will also contain restriction endonuclease sites that allow the removal of the plasmid sequences prior to transformation. The present invention will be more readily un- derstood by referring to the following example which is given to illustrate the invention rather than to limit its scope. EXAMPLE I Recombination of foreign DNA with homologous terminating ends following microinjection into bovine embryos The annealing pathway of homologous recombina¬ tion (HR) appears to be functional in a number of higher eukaryotic systems, including mammalian embryos. In accordance with this Example, the efficiency of the process in fertilized bovine oocytes was studied. The possibility of improving HR transgene incorporation reported by Brinster and colleagues was examined (1989, Proc . Na tl . Acad. Sci . USA, 86:7087) in flanking the 3' only or both ends of the foreign marker DNA with halves of a bovine Satellite DNA. This centromeric sequence was estimated as existing in at least a thousand copies in the genome. A high frequency of recombination dur¬ ing the S phase was observed in centromeric regions of Drosophila genomes. Gene targeting in the bovine satellite DNA sequences was performed by microinjection in interpha- sic zygotes (Gagne et al. , 1995, Mol . Reprod. Dev. , 41:184-194) for the following reasons: 1) to improve transgenesis' efficiency in bovine species, 2) to pre¬ vent mosaisism resulting from transgenes introduced by microinjection into eggs that are not usually repre¬ sented in the germ line of the first generation: 3) to drive the integration of foreign DNA in such a way to control the influence of the regions around the inte¬ gration site and to prevent the alteration of a vital gene by insertion: 4) to allow selection of embryos showing site specific integration by the PCR technique before transfer into recipient cows. Preparation of Bovine Oocvtes Bovine ovaries were obtained from a local slaughterhouse. Cumulus-oocyte complexes were aspirated from antral follicles (1-5 mm in diameter) with a hypo- dermic needle (18 G), selected for a compact and com¬ plete cumulus, and then washed three times in tyrode- Hepes medium supplemented with 3 mg/ml fatty acid free bovine serum albumin (BSA), 0.2 mM pyruvate, 50 μg/ml gentamicin, 5 mM glucose, and pH adjusted to 7.4 before use. Oocvte Maturation (IVM) and Fertilization (IVF) Maturation of groups of 10 cumulus-enclosed oocytes per 50 μl drop of culture medium was carried out in 60-mm petri dishes for 25 h at 38.5°C in 5% CO2 and air with water-saturated atmosphere. Oocyte matura- tion medium consisted of TCM 199 supplemented with 10% heat-treated fetal calf serum (FCS: Gibco Lab, NY), 2.2 mg/ml NaHC03, 5 μg/ml of oLH, NIADDK (National Institute of Diabetes and Digestive and Kidney Dis- eases), 0.5 μg/ml NIADDK oFSH-17, 1 μg/ml estradiol-17fl (Sigma Co, MO), 0.2 mM pyruvate, and 50 μg/ml gentami¬ cin. In vi tro fertilization was carried out as fol¬ lows. Two μl of swim-up-separated semen was added to the oil-covered fertilization drops containing five matured oocytes for a final concentration of 1 x 10^ cells/ml. Fertilization medium consisted of tyrode lac- tate medium supplemented with 0.6% BSA, 0.2 mM pyru¬ vate, 2 μg/ml heparin, and 50 μg/ml gentamicin. Fer¬ tilization was performed at 38.5°C in 5% CO2 in water- saturated atmosphere. The same procedure can be applied to in vivo matured oocytes or alternatively zygotes can be obtained after fertilization in vivo . DNA Constructs and Microinjection Construction were performed according to stan¬ dard DNA recombination procedures (Maniatis et al., 1982, Molecular Cloning, Cold Spring Harbor). Fig. 2 illustrates the construct synthesis, where PCR ampli¬ fied bovine satellite sequence (1.3 kb) was integrated in a Sma I (S) digested pPoly III vector (2.1 kb). A Sal I (Sa) fragment (215 bp) was removed from the plas¬ mid pPIII-BS(3.5 kb), and the resulting Sal I pPIII-BS (3.285 kb) was directly injected as one-sided homolo¬ gous DNA. For the other construct, a Pvu II (P) frag- ment of the Neo r gene (620 bp) was ligated to the Stu I (St) cleaved pPIII-BS. The fragment BS-Neo (1.54 kb) was obtained by digestion of the plasmid pPIII-BS-Neo- 620 with Ban II (B) . Bovine satellite sequences (BS) were used as the homologous region. Bovine genomic DNA was extracted, purified and the 1.3 kb BS sequence was amplified by PCR. Oligonucleotide primers are shown in Fig. 3. The PCR product was electrophorized on a 1% agarose gel, and extracted from the gel by electrodialysis. The amplified BS sequence was introduced in the plasmid vector pPoly III (2.1 kb) digested at the Sma I site located in the polylinker. The resulting pPIII-BS plasmid vector (3.4 kb) was opened in the BS sequence at the unique Stu I site (near the center of the sequence), or at the Sal I site (located at the 3' end of the BS sequence). The Sal I cleaved pPIII-BS plas¬ mid was used directly for microinjection. A segment of 620 bp of the Neo r gene (Neo-620) was isolated from the plasmid pMCl-Neo (Stratagene, La Jolla, CA, USA) by digestion with restriction enzyme Pvu II. The Pvu II Neo-620 fragment was than ligated with the Stu I digested pPIII-BS plasmid. The BS-Neo fragment (1.532 kb) was finally excised from the pPIIl-BS-Neo-620 con¬ struct by digestion with Ban II. The linearized Sal I pPIII-BS and the BS-Neo fragment were injected into one of the bovine oocytes pronuclei between 17 and 19 h post-insemination (hpi). This timing was chosen because this is the point at which the chromatin repli¬ cation process is at its maximum rate and the embryos are most resistant to microinjection (Gagne et al., 1995, Mol . Reprod. Dev. , 41:184-194). DNA fragments were injected at concentrations of 0.1, 0.5, 1.0 and 2.0 ng/μl, diluted in a T10E0.5 (Tris 10 mM, EDTA 0.5 mM, pH 7.4) buffer. It was verified whether this plas¬ mid had any effect on the normal development of embryos by comparing with results of injection of the pRGH-527 plasmid which does not contain homologous regions at 2 ng/μl. In vi tro Development and Culture of Embryos In vi tro development of embryos took place in 50 μl drops of TCM-199, supplemented with 10% FCS, 0.2 mM pyruvate, and 50 μg/ml gentamicin, under oil, at 38.5°C, 5% C02 an air with a water saturated atmos¬ phere. Embryos were co-cultured with bovine epithelial oviductal cells as described before (Gagne at al., 1991, Mol . Reprod. Dev. , 29:6). Culture medium was replenished after 2 days by adding 50 μl of fresh medium. After 5 days of culture, the development of embryos was assessed on the basis of morphological cri¬ teria. DNA amplification After 5 days of culture, whole embryos were extracted from zona pellucida by dissection with a 25 G needle in PBS (Ca 2+ -Mg 2+ free), washed once taking care of changing the pipette at each manipulation. The embryonic cells were placed in a 100 μl sterile reac¬ tion mixture consisting of 10 mM Tris-HCl, pH 8.3, 50 mM KC1, 1.5 mM MgCl2 and 20 mM of each deoxynucleo- tide. For the first amplification, a set of primers (Figs. 2-3) was chosen to detect the homologous recom¬ bination. For the pPIII-BS plasmid (one side homol¬ ogy), one primer started at the 3' end of the pPIIl sequence (oligo pPI I-1), and in the case of the BS-Neo fragment (two sides homology), one primer started at the 3' end of the Neo sequence (oligo Neo), and the complementary primer for both amplifications started at the 3' end of the genomic BS sequence (oligo BS). A control set of primers was used to identify the genomic DNA by amplifying a 320 bp targeted sequence of the Kappa-casein gene (oligos kappa 1 and 2). A second amplification set to detect the presence of the com¬ plete pPIII-BS-Neo-620 or uncleaved pPIII-BS plasmids (which could allow amplification and thus give a false HR positive response) was carried out on 10 ul of each resulting PCR product. Amplification with primers pPIII-2 and -3 should give a band of 420 bp on the gel if one of the plasmids is present in the samples. Oligonucleotide primer sequences: PIII-1 (SEQ. ID. NO:l) 5 ' -GGGTATCACGAGGCCGGAT-3 * pPIII-2 (SEQ. ID. NO:2) 5 ' -GGCACCTATCTCAGCGAT-3 ' pPIII-3 (SEQ. ID. NO:3) 5 '-GGAGGACCGAAGGAGCT-3 ' Neo (SEQ. ID. NO:4) 5 ' -CCGCCTGGGTGGAGAGGCTATT-3 ' BS (SEQ. ID. NO: 5) 5 ' -CCGTCATCGCAAGATGAAGCCCT-3 ' KC-1 (SEQ. ID. NO:6) 5 '-CCTGCCCAAATTCCTCAATGG-3 ' KC-2 (SEQ. ID. NO:7 ) 5 'CTGCGTTGTCTTCTTTGATGTC-3 ' The complete plasmid pPIII-BS-Neo-620 was used to determine the appropriate amplification conditions. Before adding 2.5 units of Taq DNA polymerase (Perkin- Elmer Cetus) , the samples were heated for 10 min at 100°C. The solution was covered with 100 μl of mineral oil, and amplification was carried out using a Perkin- Elmer Cetus thermal cycler. The procedure consisted of 40 cycles of denaturation at 93°C for 30 sec, anneal- ing at 59°C for 30 s and extension at 72°C for 90 sec. To avoid false-positive detections, which result mainly from contamination of the sample with the final ampli¬ fication product, we used several measures including physical isolation of the PCR preparation from the final products and use of positive displacement pipettes (Gilson, Villiers-Le-Bel France). Following amplification, 20 μl of each sample were submitted to electrophoresis on a 1% agarose gel. Statistical analysis was carried out by Chi-square test. Effects of Microin-iected constructs pPIII-BS and BS-Neo on the In vi tro development of embryos Linearized pPoly III plasmid vectors were injected in 18 hpi bovine oocytes. They were flanked at the 3' end by a 1.3 kb bovine satellite (BS) sequence, and a 620 bp fragment of the Neo gene flanked at each side respectively by 5 ' and 3 ' halves of the BS sequences. A group of untreated zygotes was kept as control and a group was injected with the Bgl II lin¬ earized plasmid pRGH-527 as foreign DNA without homolo¬ gous sequences (nHS). From the present experiments, we observed that injection of the nHS plasmid signifi¬ cantly affected (p < 0.005) embryo development : 12% (n = 53) of treated embryos and 23% (n = 278) of controls developed to morula stage (> 16 cells). Surprisingly, when injected foreign DNA was flanked by homologous sequences on one or both sides, embryo development was even more impaired. Zygotes in which 0.1, 0.5, 1.0 and 2.0 ng/μl of pPIII-BS (one-sided homology) were injected reached the morula stage in proportions of 9% (n = 77), 6% (n = 116), 4% (n = 95), and 1% (n = 75) respectively. Embryos in which BS-Neo (two-sided homology) was injected, have developed to morula stage in proportions of 11% (n = 92), 4% (n = 104), 6% (n = 104), and 0% (n = 88) respectively. The harmful effects of homologous sequences on embryo development was significantly higher (p < 0.01) at concentrations as low as 0.5 ng/μl than in nHS pRGH-527 plasmid at concentrations of 2 ng/μl. Evidence of genomic insertion with One-Side and Bilat- eral Homologous transgene The basic strategy used here was to select prim¬ ers such that amplification could occur only when homologous recombination had taken place. As illus¬ trated in Fig. 3, primers pPIII-1 and Neo were chosen from the region unique to the injected constructs and primer BS from genomic DNA sequence in the targeted gene but outside of the region used for the HR con¬ structs. Amplification products should be produced only when a construct carrying homologous extremities has been incorporated in its chromosomal homologous sequence and the primers pPIII-1 and Neo sequences have thus been integrated in continuity with the primer BS genomic sequence; no exponential amplification products should appear when the HR constructs have failed to be integrated or have been integrated at a random genomic site. If integration of injected pPIII-BS and BS-Neo DNA had occurred at the homologous genomic sequences, we should have detect the 1.5 kb and 750 bp fragments respectively on the agarose gels. The genomic control kappa-casein (KC) primers should give a fragment of 320 bp. In Fig. 4, PCR assay to detect the one-sided HR was performed on 11 embryos developed beyond the 12- cell stage. Of the 11 embryos tested, 3 were found PCR positive for the HR event because of the presence of the 1.5 kb product (lanes 3, 6 and 7). When homologous sequences were presented at both sides of the foreign DNA, which was the case for the BS-Neo fragment, the 750 bp band specific to HR was detected in 12 embryos (lanes 3, 5, 6, 9, 10, 11, 14, 17, 18, 22, 23 and 26) out of 26 developed beyond the 12-cell stage. (Fig. 5). We are well aware that positive PCR response for HR of injected DNA could be due to the presence of uncleaved and contaminant plasmid constructs, which before digestion contain the annealing sequence of the primer BS, and might allow amplification of the tar¬ geted HR sequences. Further analysis was conducted on the PCR reaction mixture following amplification to identify whether the PCR HR-products were due to a con¬ taminating whole plasmid. Amplification was performed with primers pPIII-2 and -3 specific to a pPolylll region (420 bp) . In Fig. 5, the band of 320 bp spe¬ cific to the KC gene was present in all samples, including uninjected embryos (lane 2), but not in the negative and plasmid controls (lanes 1 and 14 respec- tively), thus indicating that genomic DNA from embryos was present. However, the targeted sequence of pPolylll was not detected in 10 of the 12 PCR samples that were HR-positive from the previous amplification. In the other two samples, (lanes 7 and 13) some weak unspecific bands were observed, including one at 420 bp. Conclusion Conventional techniques like restriction enzyme digestion and Southern blot are generally used for the molecular analysis of events such as HR. However, it was previously observed (Gagne et al. , 1991, Mol . Reprod. Dev. , 29:6) that there is not enough genomic DNA in a single bovine embryo to perform these methods. In addition, it was not possible to use the expression of a foreign gene as a recombination marker since it was also observed (Gagne et al., 1994, Transgene, 1:293) that the transcription of the E coli lacZ gene driven by the 5 ' flanking region of the chicken β-actin gene could be supported by embryonic cells without being integrated. PCR is an alternative method which has been used to reveal HR events in eukaryotic cells (Zimmer and Gruss, 1989, Nature, 338:150). Current recombination models require the pres- ence of homology between two DNA sequences on both sides of a recombining site to initiate a recombination reaction. The observation that the frequency of DNA integration is increased by addition of homologous sequence on one side in mammalian cells (Adair et al., 1989, Proc . Natl . Acad. Sci . USA, 86:4572) supports our results. In the case of the two-sided homologous con¬ struct, 38 percent of embryos retained (beyond the 12- cell stage) enabled amplification of the 750 bp frag¬ ment used to identify the HR event. In conclusion, the molecular approach is criti¬ cal in attempts to produce transgenic domestic animals. The data presented here supports that transgenesis via homologous recombination by using a highly repeated genomic sequence is possible in bovine embryos. The results demonstrates that transgenesis efficiency by homologous recombination is potentially high. Once embryonic cells are increasingly transformed by homolo¬ gous recombination with repeated sequences, they can be selected by PCR to know if the inserted DNA fragment is integrated to the genome. While the invention has been described in con¬ nection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any varia¬ tions, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims. SEQUENCE LISTING (1) GENERAL INFORMATION (i) APPLICANT: Universite Laval (ii) TITLE OF THE INVENTION: INTEGRATION CASSETTE FOR IMPROVEMENT OF TRANSGENESIS IN EUKARYOTES (iii) NUMBER OF SEQUENCES: 7 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: SWABEY OGILVY RENAULT (B) STREET: 1981 McGill College Ave. - Suite 1600 (C) CITY: Montreal (D) STATE: QC (E) COUNTRY: Canada (F) ZIP: H3A 2Y3 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: DOS (D) SOFTWARE: FastSEQ for Windows Version 2.0 ( i) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 08/656,838 (B) FILING DATE: 03-JUN-1996 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Cόte, France (B) REGISTRATION NUMBER: 4166 (C) REFERENCE/DOCKET NUMBER: 6013-30PCT FC/ld (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 514 845-7126 (B) TELEFAX: 514-288-8389 (C) TELEX: (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: GGGTATCACG AGGCCGGAT 19 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GGCACCTATC TCAGCGAT 18 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GGAGGACCGA AGGAGCT 17 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: : CCGCCTGGGT GGAGAGGCTA TT 22 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: CCGTCATCGC AAGATGAAGC CCT 23 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CCTGCCCAAA TTCCTCAATG G 21 (2) INFORMATION FOR SEQ ID NO: : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CTGCGTTGTC TTCTTTGATG TC 22

Claims: WHAT IS CLAIMED IS: 1. A DNA construct for inserting a DNA fragment of interest into eukaryotic host cell, said construct com¬ prising an integration cassette flanked by site-spe¬ cific recombination sequences in which is inserted said DNA of interest, wherein said DNA fragment of interest is flanked by a nucleotide sequence sharing homology to a nucleotide sequence present in more than one copy in the eukaryotic cell, whereby said integration cassette improves the genomic insertion of said DNA fragment of interest in a site-specific manner. 2. The DNA construct of claim 1, wherein said DNA sequence of interest is flanked by nucleotide sequences sharing homology to a repeated nucleotide sequence pre¬ sent in the eukaryotic cell, wherein the flanking nucleotide sequence being linked to only one extremity or to both extremities of a DNA fragment of interest to be integrated into a host cell's genome. 3. A DNA construct of claim 2, wherein the flanking nucleotide sequences sharing homology with a DNA sequence of the host cell's genome is selected from the group consisting of a Satellite DNA sequence, cortico- tropin-β-lipotropin, non-satellite repetitive DNA, his- tone, ribosomic RNA transfer RNA coding sequences, Pst, Bsu and Alu-like repetitive sequences. 4. A DNA construct of claim 3, wherein the DNA of interest is linked to regulatory sequences capable of expressing the said DNA of interest in the eukaryotic cell. 5. A DNA construct of claim 1, which further com¬ prises DNA sequences capable of replicating the DNA molecule in a bacterial host, and DNA sequences encod¬ ing a bacterial selectable marker. 6. A DNA construct of claim 4, wherein a DNA sequence of interest can be transcribed into a messen¬ ger RNA for production of an anti-mRNA, ribosomes or after translation of the said RNA, for production of a protein of interest. 7. A DNA construct of claim 1, wherein at the 5' extremity is linked a nucleotidic fragment of 2 to 10^ base pairs sharing homology with endogenous sequences existing into the host cell's genome, where said flank¬ ing sequences are repeated from about two times to about one million times. 8. A DNA construct of claim 1, wherein at the 3' extremity is linked a nucleotide fragment of 2 to 10^ base pairs sharing homology with repeated sequences existing into the host cell's genome. 9. A DNA construct of claim 1, wherein at the 5' and 3 ' extremities of the same DNA construct to be integrated into a host cell's genome, are linked nucleotide fragments of 2 to 10^ base pairs sharing homology with repeated sequences existing in the host cell's genome. 10. A DNA construct of claim 9, wherein homology of the 5 ' and the 3 ' flanking nucleotide fragments can be partial or complete with the nucleotide sequences existing into the host cell's genome, and said flanking DNA sequences. 11. A DNA construct of claim 10, wherein said nucleotide sequences shares homology with repeated sequences existing in the host cell's genome, where said nucleotide sequences are repeated on both extremi¬ ties of a DNA fragment of interest to be integrated to the host cell's genome, or parts of said nucleotide sequences are linked at each extremities of the said DNA fragment of interest. 12. Use of a DNA construct of claim 2 to increase the rate of insertion of a DNA sequence of interest into the DNA of a eukaryotic cell. 13. A method for improvement of the production of fertile, transgenic eukaryotic organisms wherein the transgenic eukaryotic organisms have a DNA sequence of interest integrated at a predetermined repeated DNA sequence of the organisms, said method comprising the steps of introducing into eukaryotic cells a DNA con¬ struct comprising a gene of interest flanked by site- specific recombination sequences, wherein gene of interest is flanked by nucleotide sequences sharing homology to the predetermined nucleotide sequence pre¬ sent in the eukaryotic cell, and said gene of interest is operably linked to regulatory sequences capable of expressing the gene in the eukaryotic cell. 14. A method of claim 13, wherein the eukaryotic organisms are diploid plant and animal cells. 15. A method of directly selecting for insertion of a DNA sequence of interest into a specific sequence of an organism's DNA said method comprising the steps of a) introducing a DNA construct of claim 1 into the cells of said organisms; b) choosing a pair of primers, wherein one primer recognizes a DNA sequences of the DNA fragment of interest, and the other primer recognizes a DNA sequence of the host cell, where said sequence recognized by the said other primer exists outside of the DNA construct of claim 1; c) using said pair of primers of step b) to amplify the said DNA construct inserted into the host cell's genome by homologous recombination; and d) selecting those cells having said DNA construct integrated into the DNA of the cell, thus being transgenic cells.

Inventors: GAGNE MARC, POTHIER FRANCOIS, SIRARD MARC-ANDRE, GAGNE, MARC, POTHIER, FRANCOIS, SIRARD, MARC-ANDRE
Applicants: UNIV LAVAL, UNIVERSITE LAVAL
Application year: 1997
Main classification: C12N15
IPC codes: C12N15