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. 1997 Oct;71(10):7820–7826. doi: 10.1128/jvi.71.10.7820-7826.1997

Development of high-titer retroviral producer cell lines by using Cre-mediated recombination.

E F Vanin 1, L Cerruti 1, N Tran 1, G Grosveld 1, J M Cunningham 1, S M Jane 1
PMCID: PMC192136  PMID: 9311869

Abstract

Retroviral gene transfer is widely used in experimental and human gene therapy applications. We have devised a novel method of generating high-titer retroviral producer cell lines based on the P1 bacteriophage recombinase system Cre-loxP. Incorporation of loxP sites flanking a Neo(r)-SVTK cassette in the proviral DNA allows excision of these selectable markers through expression of Cre recombinase after production of a high-titer producer cell line. The resultant producer line contains a single loxP site flanked by the viral long terminal repeats. Retransfection of this line with the Cre expression vector and a plasmid containing a gene of interest flanked by loxP sites allows insertional recombination of the gene into the favorable preexisting site in the genome and the generation of a new line with a titer equivalent to that of the parental producer cell line. The efficiency of the process is sufficient to allow the generation of multiple new producer lines without the addition of antibiotic resistance genes. We have successfully generated retroviral vectors carrying different genes by using this approach and discuss the potential applications of this method in gene therapy.

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Selected References

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  1. Apperley J. F., Luskey B. D., Williams D. A. Retroviral gene transfer of human adenosine deaminase in murine hematopoietic cells: effect of selectable marker sequences on long-term expression. Blood. 1991 Jul 15;78(2):310–317. [PubMed] [Google Scholar]
  2. Austin S., Ziese M., Sternberg N. A novel role for site-specific recombination in maintenance of bacterial replicons. Cell. 1981 Sep;25(3):729–736. doi: 10.1016/0092-8674(81)90180-x. [DOI] [PubMed] [Google Scholar]
  3. Bergemann J., Kühlcke K., Fehse B., Ratz I., Ostertag W., Lother H. Excision of specific DNA-sequences from integrated retroviral vectors via site-specific recombination. Nucleic Acids Res. 1995 Nov 11;23(21):4451–4456. doi: 10.1093/nar/23.21.4451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bertran J., Miller J. L., Yang Y., Fenimore-Justman A., Rueda F., Vanin E. F., Nienhuis A. W. Recombinant adeno-associated virus-mediated high-efficiency, transient expression of the murine cationic amino acid transporter (ecotropic retroviral receptor) permits stable transduction of human HeLa cells by ecotropic retroviral vectors. J Virol. 1996 Oct;70(10):6759–6766. doi: 10.1128/jvi.70.10.6759-6766.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bowtell D. D., Cory S., Johnson G. R., Gonda T. J. Comparison of expression in hemopoietic cells by retroviral vectors carrying two genes. J Virol. 1988 Jul;62(7):2464–2473. doi: 10.1128/jvi.62.7.2464-2473.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Choulika A., Guyot V., Nicolas J. F. Transfer of single gene-containing long terminal repeats into the genome of mammalian cells by a retroviral vector carrying the cre gene and the loxP site. J Virol. 1996 Mar;70(3):1792–1798. doi: 10.1128/jvi.70.3.1792-1798.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Crystal R. G. Transfer of genes to humans: early lessons and obstacles to success. Science. 1995 Oct 20;270(5235):404–410. doi: 10.1126/science.270.5235.404. [DOI] [PubMed] [Google Scholar]
  8. Danos O., Mulligan R. C. Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6460–6464. doi: 10.1073/pnas.85.17.6460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dougherty J. P., Wisniewski R., Yang S. L., Rhode B. W., Temin H. M. New retrovirus helper cells with almost no nucleotide sequence homology to retrovirus vectors. J Virol. 1989 Jul;63(7):3209–3212. doi: 10.1128/jvi.63.7.3209-3212.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gossen M., Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5547–5551. doi: 10.1073/pnas.89.12.5547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gu H., Zou Y. R., Rajewsky K. Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting. Cell. 1993 Jun 18;73(6):1155–1164. doi: 10.1016/0092-8674(93)90644-6. [DOI] [PubMed] [Google Scholar]
  12. Hoess R. H., Abremski K. Interaction of the bacteriophage P1 recombinase Cre with the recombining site loxP. Proc Natl Acad Sci U S A. 1984 Feb;81(4):1026–1029. doi: 10.1073/pnas.81.4.1026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Julias J. G., Hash D., Pathak V. K. E- vectors: development of novel self-inactivating and self-activating retroviral vectors for safer gene therapy. J Virol. 1995 Nov;69(11):6839–6846. doi: 10.1128/jvi.69.11.6839-6846.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Karreman S., Hauser H., Karreman C. On the use of double FLP recognition targets (FRTs) in the LTR of retroviruses for the construction of high producer cell lines. Nucleic Acids Res. 1996 May 1;24(9):1616–1624. doi: 10.1093/nar/24.9.1616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kasahara N., Dozy A. M., Kan Y. W. Tissue-specific targeting of retroviral vectors through ligand-receptor interactions. Science. 1994 Nov 25;266(5189):1373–1376. doi: 10.1126/science.7973726. [DOI] [PubMed] [Google Scholar]
  16. Loh T. P., Sievert L. L., Scott R. W. Proviral sequences that restrict retroviral expression in mouse embryonal carcinoma cells. Mol Cell Biol. 1987 Oct;7(10):3775–3784. doi: 10.1128/mcb.7.10.3775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Markowitz D., Goff S., Bank A. Construction and use of a safe and efficient amphotropic packaging cell line. Virology. 1988 Dec;167(2):400–406. [PubMed] [Google Scholar]
  18. Miller A. D. Human gene therapy comes of age. Nature. 1992 Jun 11;357(6378):455–460. doi: 10.1038/357455a0. [DOI] [PubMed] [Google Scholar]
  19. Miller A. D. Retroviral vectors. Curr Top Microbiol Immunol. 1992;158:1–24. doi: 10.1007/978-3-642-75608-5_1. [DOI] [PubMed] [Google Scholar]
  20. Miller A. D., Rosman G. J. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989 Oct;7(9):980-2, 984-6, 989-90. [PMC free article] [PubMed] [Google Scholar]
  21. Parks R. J., Chen L., Anton M., Sankar U., Rudnicki M. A., Graham F. L. A helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):13565–13570. doi: 10.1073/pnas.93.24.13565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Reddy S., DeGregori J. V., von Melchner H., Ruley H. E. Retrovirus promoter-trap vector to induce lacZ gene fusions in mammalian cells. J Virol. 1991 Mar;65(3):1507–1515. doi: 10.1128/jvi.65.3.1507-1515.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Russ A. P., Friedel C., Grez M., von Melchner H. Self-deleting retrovirus vectors for gene therapy. J Virol. 1996 Aug;70(8):4927–4932. doi: 10.1128/jvi.70.8.4927-4932.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sauer B. Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1987 Jun;7(6):2087–2096. doi: 10.1128/mcb.7.6.2087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sauer B., Henderson N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5166–5170. doi: 10.1073/pnas.85.14.5166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sauer B. Manipulation of transgenes by site-specific recombination: use of Cre recombinase. Methods Enzymol. 1993;225:890–900. doi: 10.1016/0076-6879(93)25056-8. [DOI] [PubMed] [Google Scholar]
  27. Sauer B., Whealy M., Robbins A., Enquist L. Site-specific insertion of DNA into a pseudorabies virus vector. Proc Natl Acad Sci U S A. 1987 Dec;84(24):9108–9112. doi: 10.1073/pnas.84.24.9108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tsichlis P. N., Lazo P. A. Virus-host interactions and the pathogenesis of murine and human oncogenic retroviruses. Curr Top Microbiol Immunol. 1991;171:95–171. doi: 10.1007/978-3-642-76524-7_5. [DOI] [PubMed] [Google Scholar]
  29. Valera A., Perales J. C., Hatzoglou M., Bosch F. Expression of the neomycin-resistance (neo) gene induces alterations in gene expression and metabolism. Hum Gene Ther. 1994 Apr;5(4):449–456. doi: 10.1089/hum.1994.5.4-449. [DOI] [PubMed] [Google Scholar]
  30. Vanin E. F., Kaloss M., Broscius C., Nienhuis A. W. Characterization of replication-competent retroviruses from nonhuman primates with virus-induced T-cell lymphomas and observations regarding the mechanism of oncogenesis. J Virol. 1994 Jul;68(7):4241–4250. doi: 10.1128/jvi.68.7.4241-4250.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Varmus H. Retroviruses. Science. 1988 Jun 10;240(4858):1427–1435. doi: 10.1126/science.3287617. [DOI] [PubMed] [Google Scholar]
  32. Westerman K. A., Leboulch P. Reversible immortalization of mammalian cells mediated by retroviral transfer and site-specific recombination. Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):8971–8976. doi: 10.1073/pnas.93.17.8971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wilson C., Reitz M. S., Okayama H., Eiden M. V. Formation of infectious hybrid virions with gibbon ape leukemia virus and human T-cell leukemia virus retroviral envelope glycoproteins and the gag and pol proteins of Moloney murine leukemia virus. J Virol. 1989 May;63(5):2374–2378. doi: 10.1128/jvi.63.5.2374-2378.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yang Y., Vanin E. F., Whitt M. A., Fornerod M., Zwart R., Schneiderman R. D., Grosveld G., Nienhuis A. W. Inducible, high-level production of infectious murine leukemia retroviral vector particles pseudotyped with vesicular stomatitis virus G envelope protein. Hum Gene Ther. 1995 Sep;6(9):1203–1213. doi: 10.1089/hum.1995.6.9-1203. [DOI] [PubMed] [Google Scholar]
  35. von Melchner H., Housman D. E. The expression of neomycin phosphotransferase in human promyelocytic leukemia cells (HL60) delays their differentiation. Oncogene. 1988 Feb;2(2):137–140. [PubMed] [Google Scholar]

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