SYSTEM FOR AMPLIFICATION OF EUKARYOTIC GENES

Abstract: Amplification of eukaryotic genes in mammalian cells by linking the genes to CAD genes so that each cell contains multicopy numbers of the linked genes. The product of the expression of the eukaryotic genes may then be recovered from the mammalian cells.

Description: SYSTE FOR AMPLIFICATION OF EUKARYOTIC GENES This invention relates generally to recombinan DNA technology and, more particularly, to improvements whereby the product of the expression of eukaryotic genes is produced in large amounts in mammalian cells. As is well known, the particular sequence of a ino acids in a given protein is determined in accordanc with the code carried in the gene for that protein. In the process of expressing the protein which it encodes, through the KNA intermediary, groups of three nucleotides in the DNA, called codons , each place one of 20 possible amino acids at a corresponding position in the protein chain. There are four possible nucleotides and,, in the normal double stranded DNA, each nucleotide in one strand is adjacent a complimentary nucleotide in the other strand. With the advent of recombinant DNA techniques , genetic changes may be made deliberately by the intro¬ duction of a predetermined nucleotide sequence, either synthesized or isolated from one strain or species, into the genetic makeup of another strain or species . The strain or species into which the recombinant sequence is introduced produces, as part of its normal processes, the protein encoded by the newly introduced DNA. When the modified strain or species proceeds with the normal repli¬ cation process, it also duplicates the inserted sequence. Various techniques are known for isolating a desired nucleotide sequence or gene from one species, or constructing that sequence synthetically. In addition, there are many known techniques for introducing the thus isolated gene into the genetic system of a desired host. Such techniques include the utilization of plasmids or phages which are broken open by restriction enzymes to allow the insertion of the isolated gene. Such plasmids or phages are then reintroduced to a suitable bacterial host species , such as Ξ. coli, where they are ' capable of being replicated and wherein the protein for which they encode is expressed. Thus far, much of the work in the area of recombinant DNA has involved strains of E. coli or some other bacterial strain, into the genetic system of which has been introduced a desired eukaryotic gene. Examples include the genes for human growth hormone, insulin, and interferon. Although potentially very useful for produc¬ ing otherwise scarce biologically important substances, the use of bacterial cells also has inherent drawbacks. For example, typically only trace quantities of the desired substances are produced by known bacterial strains. In some cases, further processing of the protei products of the bacteria is necessary in order to isolate the desired molecule which often occurs in low yields. Finally, the use of bacteria for the production of sub¬ stances which are medically useful for humans raises questions regarding safety for clinical use. It is as yet unsettled as to how extensive the required tests will be in order to obtain approval from the appropriate gov- ernmental agencies for use of such substances. Accordingly, it is an object of the present invention to provide a method for producing a desired polypeptide in amplified amounts in mammalian cells. Another object of the invention is to provide an improved mammalian cell suitable for use in a process wherein living cells are used for replication and expres¬ sion of DNA segments of interest. A more general object of the invention is to provide an improved method for the production and recovery of eukaryotic gene products. Very generally, the method of the invention results in the production of the desired polypeptide in amplified amounts in mammalian cells. Genes for the de- sired polypeptide are isolated and are linked to CAD genes. The genes are transfected to mammalian cells and those of the mammalian cells having functional copies of both linked genes are selected. , These selected cells are further selected for resistance to substantial levels of PALA. The desired polypeptide may then be recovered from the further selected PALA-resistant cells in ampli¬ fied amounts. The present invention takes advantage of a peculiar phenomenon respecting the resistance of mammalia tissue culture cells to PALA, a transition state analog inhibitor of aspartate transcarbamylase, one of the three enzymatic activities of the multifunctional protein CAD. PALA is ' otherwise known as N- (phosphonacetyl) -L- aspartate. CAD catalyzes the first three reactions of de_ novo UMP biosynthesis. The phenomenon referred to is that all PALA-resistant cells have amplified amounts of the CAD gene. Using sequential selection steps for resistance to increasing levels of PALA, cell lines containing 100- 200 times the number of CAD genes as the wild type cells may be obtained. (A PALA concentration at which the frequency of resistance of wild type cells is less than —8 10 is a suitable maximum or final concentration level) . The presence of the CAD gene in these very highly ampli- fied or multicopy number amounts is the only mechanism of PALA-resistance thus far detected. Most PALA-resis¬ tant lines can be grown in the absence of PALA for long periods without losing their multicopy number of the CAD gene. (1) In accordance with the invention, the entire -4- CAD gene is isolated from cells containing multicopy num bers of that gene. Preferably, such cells contain from 100-200 copies of the CAD gene. Either of three methods may be utilized for isolating the CAD gene. First, high molecular weight DNA obtained by random shearing or par¬ tial digestion with restriction endonucleases could be used. Second, by conventional molecular cloning into phage vectors (2,3) , it is possible ' to clone 20,000 base pairs (20kbp) of PALA-resistant syrian hamster DNA. The pieces may be then ligated to recreate a functional CAD gene in accordance with the known arrangement of the gen omic DNA in syrian hamster cells. A third way in which the CAD gene may be iso¬ lated is to clone a single piece of genomic DNA up to 40 kbp long using cosmid vectors. Cosmid vectors are a com bination of the cohesive (cos) ends of λphage and the plasmid PBR 322. These cosmids can be packaged as phage due to the locations of the cohesive ends of the vector. Recombinant clones may be selected as ampicillin or tetr cycline resistant bacteria because of the plasmid, which contains this marker (4,5) . Functionality of the cloned gene may be ascertained by determining if transfection of CAD cells (6) produces CAD transformants. The eukaryotic DNA of interest is then linked t the CAD gene. The gene of interest may be, for example, a gene for a human hormone isolated from natural cell material, or may be a gene synthesized by a suitable nuc- leotide synthesis technique (7) . One of two techniques may be utilized to achieve the linking. A suitable host cell may be co-transfected with the CAD gene and the gene of interest in a manner which results in cells containing linked arrangements of both genes. For example, Wigler and Axel (8) have shown that co-transfection of mouse -5- cells with the herpes TK gene and the bacterial virus ΦX174 results in transformants containing both DNA mole¬ cules in an apparently linked arrangement. Alternatively the CAD gene may be ligated to the desired gene either at convenient natural restriction sites or at synthetic res¬ triction sites created in vitro (1) . The linked DNA is then transfected in the linked form to the desired host b suitable known techniques.. Following the linking and transfection steps , the mammalian cells into which the linked genes have been inserted are grown and suitable techniques are utilized t select those cell cultures which exhibit the characteris¬ tics of having functional copies of both the linked genes. Such a determination may be made utilizing conventional blotting technology. The resultant selected cells thereb are capable of replication of bo.th of the linked genes an are also capable of expression of the product of those genes. A further selection is then made to select those of the previously selected mammalian cells which have a resistance to substantial levels of PALA. The gene of in¬ terest will have been co-amplified along with the CAD gene in at least some of the cells. Accordingly, the product of the co-amplified gene is produced at high levels. The desired polypeptide produced by the desired eukaryotic gene may then be recovered from the selected PALA-resis¬ tant cells. The following examples are set forth to illus- trate in greater detail the manner by which the invention is carried out. These examples are not intended to limit the scope of the claims. Example I A PALA-resistant cell line with approximately 100-200 CAD genes per cell (1) is processed by known methods to isolate the DNA. The DNA is then digested partially with endonuclease EcoRl following standard procedures. This partially digested DNA is then fractionated into fragments approximately 25-40 kbp in length using sucrose gradient centrifugation. The 25-40 kbp fragments are the ligated to an appropriate EcoRl digested cosmid (e.g., MUA3 of Meyerowitz et al., 5). The cosmid is- then pack¬ aged in vitro into the heads of ba ' cteriophage lambda (4) . To minimize the occurrence of deletion, the cos ids are then used to "infect" a recA~ * host such a HB101. Trans- formants containing the cosmids with the CAD gene inserts are located using the colony filter or plaque filter hy¬ bridization techniques (9,10) . Those cosmids with CAD sequences are selected and are analyzed further by restriction enzyme analysis using- probes for the 5' and 3' proximal regions of a 19 kbp EcoRl CAD clone. This clone has been fully character ized ' (1) - Those clones with inserts extending to the 5 ! end and 3 ' ends of the 19 kbp EcoRl CAD fragment are then tested for functionality using the calcium phosphate transfection technique for mammalian cells (11,12) . Co-transfection of mammalian cells is then effected using the CAD genes as isolated above and the gene of interest by means of the co-transfection procedur described by Wigler and Axel (9) . Conventional blotting technology is used to detect cells containing both genes and a further selection for PALA-resistant cells is made. Those PALA-resistant cells which indicate co-amplificatio of the gene of interest are then assessed by RNA (13) and DNA (14) blotting experiments. Radioimmune assays, e.g. (15) , to determine whether the correct protein is produce are then conducted and an estimate of the increase in the amount of expression by the desired gene may be made on the basis of the selection for different levels of PALA resistancε. Example II The procedures of Example I are followed except that, in order to infect the bacterial host with CAD gene for purification, a pseudophage vector is used such as de signed by Alton and Davis (16) . The latter vector has th advantage of selecting against recombinants which have un dergone deletion events in the process of replication in bacterial host. Example III The procedure of Example I is followed except that in isolating the CAD genes, microcells (17) are used as vehicles for introducing the large DNA molecules (i.e. multicopy CAD genes) to mammalian cells in culture. Example IV The procedures of Example I are followed except that in isolating the CAD genes, synthetic lipid vesicles (18) are used as vehicles for introducing the large DNA molecules (i.e. CAD genes) to mammalian cells in culture. Example V A procedure identical with that of Example I is followed except that mechanical microinjection is used to insert the large DNA molecules. The advantages of the foregoing procedures are, basically, two-fold. First, it is possible to amplify any gene in any mammalian cell of interest using PALA-resis- tance as the selective marker. By using transfecting DNA obtained from cells in which it is present initially as many copies, it is unnecessary to utilize genetically marked cells, in effect, CAD minus cells. The second basic advantage provided by the fore¬ going technique is that introduction and amplification of the desired gene in a cell which normally produces and/or exports the product of that gene will typically insure that the product is processed correctly. Thus, further processing or modification of the gene product is unneces sary in order to form the active molecule. A further advantage, of course, results from th fact that the substances are produced in mammalian tissue which may provide significant advantages from the stand¬ point of obtaining approval for use of the substances fro government agencies. It may be seen, therefore, that the invention provides an improved process for producing a desired poly peptide in amplified amounts in mammalian cells. The re¬ sultant recombinant DNA containing cells produced in accordance with the invention contain multiple copies of functional gene pairs, each comprising a CAD gene linked to the DNA segment of interest. Various modifications of the invention in addi¬ tion to those shown and described herein will be apparent to those skilled in the art from the foregoing descrip- tion. Such modifications are intended to fall within the scope of the appended claims.

Claims: _ g _ Claims ___ 1. A method for producing a desired polypeptid in amplified amounts in mammalian cells, which method comprises isolating genes for the desired polypeptide, linking said isolated genes for the desired polypeptide to CAD genes isolated from multi-copy number cells and effecting transfection to mammalian cells, growing said mammalian cells, selecting those of said mammalian cells having functional copies of both linked genes, and recovering the desired polypeptide from the selected PALA resistance cells. 2. A method according to Claim 1 wherein those of said selected mammalian cells are further selected to obtain cells having resistance to substantial levels of PALA. 3. A method according to Claim 1 or 2 wherein the genes are linked by co-transfecting genes into the mammalian cells. 4. A method according to Claim 1 or 2 wherein the genes are linked by ligating the genes prior to transfection to the mammalian cells. 5. A method according to any one of Claims 1 to 3 wherein said CAD gene is isolated by producing phages containing said CAD gene, transfecting host cells with said phages, and selecting for PALA-resistance. 6. A method according ' to any one of Claims 1 to 3 wherein the CAD gene is isolated by producing cosmid vectors containing the gene, transfecting host cells with such vectors, and selecting for PALA-resistance. 7. A method according to any one of Claims 1 to 6 wherein said multi-copy number cells from which said CAD gene is isolated have about 100-200 CAD genes. AMENDED CLAIMS (received by the Intemationai Bureau on 17 November 1981(17.11.81)) 1. A method for producing a desired polypeptid in amplified amounts in mammalian cells, which method comprises isolating genes for the desired polypeptide, linking said isolated genes for the desired polypeptide to CAD genes isolated from multi-copy number cells and effecting transfection to mammalian cells, growing said mammalian cells, selecting those of said mammalian cells having functional copies of both linked genes, and recovering the desired polypeptide from the selected PALA resistance cells. 2. A method according to Claim 1 wherein those of said selected mammalian cells are further selected to obtain cells having resistance to substantial levels of PALA. 3. A method according to Claim 1 or 2 wherein the genes are linked by co-transfecting genes into the mammalian cells. 4. A method according to Claim 1 or 2 wherein the genes are linked by ligating the genes prior to transfection to the mammalian cells. 5. A method according to any one of Claims 1 to 3 wherein said CAD gene is isolated by producing phages containing said CAD gene, transfecting host cells with said phages, and selecting for PALA-resistance. 6. A method according to any one of Claims 1 to 3 wherein the CAD gene is isolated by producing cosmid vectors containing the gene, transfecting host cells with such vectors, and selecting for PALA-resistance. 7. A method according to any one of Claims 1 to 6 wherein said multi-copy number cells from which said CAD gene is isolated have about 100-200 CAD genes. m -11- to 7 wherein said multi-copy number cells are selected f PALA-resistance using a PALA concentration at which the frequency of resistance of wild type cells is less than 10 "8 . . ( "A men d e d J 9. For use in a process wherein living cells are used for replication and expression of foreign recom nant DNA segments of interest, and wherein the product o the expression of the DNA segments of interest is re¬ covered, a mammalian cell having functional multiple copi of a CAD gene linked to the DNA segment of interest. 10. A mammalian cell according to Claim 9 containing about 100-200 CAD genes.

Inventors: WAHL G
Applicants: SALK INST FOR BIOLOGICAL STUDI, SALK INST BIOLOGICAL STUDIES
Application year: 1981
Main classification: C12N5
IPC codes: C12N5, C12N15, C12P21