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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

• 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 .
• each nucleotide in one strand is adjacent a complimentary nucleotide in the other strand.
• genetic changes may be made deliberately by the introduction 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.
• the modified strain or species proceeds with the normal replication 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.
• 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 E. coli, where they are capable of being replicated and wherein the protein for which they encode is expressed.
• 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 expression 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.
• 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 amplified amounts .
• the present invention takes advantage of a peculiar phenomenon respecting the resistance of mammalian 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.
• the entire 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 partial digestion with restriction endonucleases could be used.
• phage vectors Two,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 genomic DNA in syrian hamster cells.
• a third way in which the CAD gene may be isolated is to clone a single piece of genomic DNA up to 40 kbp long using cosmid vectors.
• Cosmid vectors are a combination of the cohesive (cos) ends of ⁇ phage 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 tetracycline 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 to 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 nucleotide synthesis technique (7) .
• 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 .
• Wigler and Axel (8) have shown that co-transfection of mouse cells with the herpes TK gene and the bacterial virus ⁇ X174 results in transformants containing both DNA molecules in an apparently linked arrangement.
• the CAD gene may be ligated to the desired gene either at convenient natural restriction sites or at synthetic restriction sites created in vitro (1) .
• the linked DNA is then transfected in the linked form to the desired host by suitable known techniques.
• the mammalian cells into which the linked genes have been inserted are grown and suitable techniques are utilized to select those cell cultures which exhibit the characteristics of having functional copies of both the linked genes. Such a determination may be made utilizing conventional blotting technology.
• the resultant selected cells thereby are capable of replication of both of the linked genes and 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 interest 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-resistant cells.
• 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 EcoR1 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 packaged in vitro into the heads of bacteriophage lambda (4) .
• the cosmids 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 hybridization 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.
• 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 proceedur 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 resistance.
• Example II The procedures of Example I are followed except that, in order to infect the bacterial host with CAD genes for purification, a pseudophage vector is used such as de signed by Alton and Davis (16) .
• the latter vector has the 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.
• 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-resistance 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 foregoing 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 unnecessary in order to form the active molecule.
• a further advantage results from the fact that the substances are produced in mammalian tissue which may provide significant advantages from the standpoint of obtaining approval for use of the substances from government agencies. It may be seen, therefore, that the invention provides an improved process for producing a desired polypeptide in amplified amounts in mammalian cells.
• the resultant 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.

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• 1981
  • 1981-07-07 JP JP50247681A patent/JPS57501112A/ja active Pending
  • 1981-07-07 WO PCT/US1981/000911 patent/WO1982000158A1/en not_active Application Discontinuation
  • 1981-07-07 EP EP19810901998 patent/EP0055742A4/de not_active Withdrawn

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