Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses
Patent
1983-10-25
1986-08-26
Wiseman, Thomas G.
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Modification of viruses
435240, 435 70, 935 93, 935 57, 935 65, 935 71, 935 73, 935 29, 935 12, C12N 1500, C12N 500, C12P 2102
Patent
active
046083391
ABSTRACT:
A modified protoplast fusion method and cell line is disclosed that stably transfects human cells with pSV2-derived plasmids at frequencies greater than 10.sup.-3. This procedure makes it possible to test the biological effect of individual genes (i.e., oncogenes and other cellular genes, and viral genes). To demonstrate the utility of this invention, a pSV2gpt.sup.+ plasmid constructed to carry a subgenomic fragment of hepatitis B virus (HBV) that contained the core antigen gene (HBc gene) is transfected into human cells. Human cell lines are stably transfected with the HBC.sup.+ gene by selecting recipient cells for expression of guanine phosphoribosyl transferase expression; other selective markers, i.e., neomycin resistance, can also be used. Conditions for enhancing the expression of the transfected gene(s) have also been developed. For example, with this gpt.sup.+ /HBc.sup.+ cell line it is shown that growth in serum-free medium or treatment with 5'-azacytidine stimulates the production of the HBV core antigen.
REFERENCES:
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Sandri-Goldin et al, "High Efficiency Transfer of DNA into Eukaryotic Cells by Protoplast Fusion", Methods in Enzymology, 101, pp. 402-411 (1983).
Sandri-Goldin et al, "High Frequency Transfer of Cloned Herpes Simplex Virus Type I Sequences to Mammalian Cells . . . ", Molecular and Cellular Biology 1(8), pp. 743-752 (1981).
Schaffner, "Direct Transfer of Cloned Genes from Bacteria to Mammalian Cells," Proceedings of the National Academy of Sciences, 77(4) pp. 2163-2167 (1980).
Rassoulzadegan et al, "High Frequency of Gene Transfer after Fusion Between Bacteria and Eukaryotic Cells", Nature, 295, pp. 257-259 (1982).
Stahl et al, "Hepatitis B Virus Core Antigen: Synthesis in Escherichia coli and Application in Diagnosis", Proceedings of the National Academy of Sciences, 79, pp. 1606-1610 (1982).
Gough et al, "Expression of Hepatitis B Virus Surface, Core and E Antigen Genes by Stable Rat and Mouse Cell Lines", Journal of Molecular Biology, 162, pp. 43-67 (1982).
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Harris Curtis C.
Korba Brent E.
Lechner John F.
Yoakum George H.
Roberts, Jr. John S.
Tarcza John E.
Wiseman Thomas G.
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