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. 1989 Jan;9(1):100–108. doi: 10.1128/mcb.9.1.100

Construction and properties of replication-competent murine retroviral vectors encoding methotrexate resistance.

H Stuhlmann 1, R Jaenisch 1, R C Mulligan 1
PMCID: PMC362150  PMID: 2927389

Abstract

A series of replication-competent Moloney murine leukemia virus vectors was constructed in which each vector contained a mutant dihydrofolate reductase (DHFR) cDNA insert in the U3 region of the viral long terminal repeat. Two of the resulting viruses, MLV (murine leukemia virus) DHFR*-5 and MLV DHFR*-7, were able to stably transfer methotrexate resistance to infected fibroblast cells upon multiple rounds of virus replication and in the absence of drug selection. Cell lines producing recombinant virus with high titers were established, which indicated that the insert did not grossly interfere with viral replication functions. These vectors should be useful for introducing and expressing foreign genes in vivo in tissues and whole animals in which virus spread is needed for efficient infection.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  2. Cepko C. L., Roberts B. E., Mulligan R. C. Construction and applications of a highly transmissible murine retrovirus shuttle vector. Cell. 1984 Jul;37(3):1053–1062. doi: 10.1016/0092-8674(84)90440-9. [DOI] [PubMed] [Google Scholar]
  3. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  4. Cone R. D., Weber-Benarous A., Baorto D., Mulligan R. C. Regulated expression of a complete human beta-globin gene encoded by a transmissible retrovirus vector. Mol Cell Biol. 1987 Feb;7(2):887–897. doi: 10.1128/mcb.7.2.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fan H., Jaenisch R., MacIsaac P. Low-multiplicity infection of Moloney murine leukemia virus in mouse cells: effect on number of viral DNA copies and virus production in producer cells. J Virol. 1978 Dec;28(3):802–809. doi: 10.1128/jvi.28.3.802-809.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Foster D. A., Hanafusa H. A fps gene without gag gene sequences transforms cells in culture and induces tumors in chickens. J Virol. 1983 Dec;48(3):744–751. doi: 10.1128/jvi.48.3.744-751.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gélinas C., Temin H. M. Nondefective spleen necrosis virus-derived vectors define the upper size limit for packaging reticuloendotheliosis viruses. Proc Natl Acad Sci U S A. 1986 Dec;83(23):9211–9215. doi: 10.1073/pnas.83.23.9211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Haber D. A., Beverley S. M., Kiely M. L., Schimke R. T. Properties of an altered dihydrofolate reductase encoded by amplified genes in cultured mouse fibroblasts. J Biol Chem. 1981 Sep 25;256(18):9501–9510. [PubMed] [Google Scholar]
  9. Hentschel C., Irminger J. C., Bucher P., Birnstiel M. L. Sea urchin histone mRNA termini are located in gene regions downstream from putative regulatory sequences. Nature. 1980 May 15;285(5761):147–151. doi: 10.1038/285147a0. [DOI] [PubMed] [Google Scholar]
  10. Hughes S. H., Greenhouse J. J., Petropoulos C. J., Sutrave P. Adaptor plasmids simplify the insertion of foreign DNA into helper-independent retroviral vectors. J Virol. 1987 Oct;61(10):3004–3012. doi: 10.1128/jvi.61.10.3004-3012.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jaenisch R. Retroviruses and embryogenesis: microinjection of Moloney leukemia virus into midgestation mouse embryos. Cell. 1980 Jan;19(1):181–188. doi: 10.1016/0092-8674(80)90399-2. [DOI] [PubMed] [Google Scholar]
  12. Jähner D., Stuhlmann H., Stewart C. L., Harbers K., Löhler J., Simon I., Jaenisch R. De novo methylation and expression of retroviral genomes during mouse embryogenesis. Nature. 1982 Aug 12;298(5875):623–628. doi: 10.1038/298623a0. [DOI] [PubMed] [Google Scholar]
  13. Kaufman R. J., Murtha P., Davies M. V. Translational efficiency of polycistronic mRNAs and their utilization to express heterologous genes in mammalian cells. EMBO J. 1987 Jan;6(1):187–193. doi: 10.1002/j.1460-2075.1987.tb04737.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kornbluth S., Cross F. R., Harbison M., Hanafusa H. Transformation of chicken embryo fibroblasts and tumor induction by the middle T antigen of polyomavirus carried in an avian retroviral vector. Mol Cell Biol. 1986 May;6(5):1545–1551. doi: 10.1128/mcb.6.5.1545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kozak M. Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes. Mol Cell Biol. 1987 Oct;7(10):3438–3445. doi: 10.1128/mcb.7.10.3438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lobel L. I., Patel M., King W., Nguyen-Huu M. C., Goff S. P. Construction and recovery of viable retroviral genomes carrying a bacterial suppressor transfer RNA gene. Science. 1985 Apr 19;228(4697):329–332. doi: 10.1126/science.2984770. [DOI] [PubMed] [Google Scholar]
  17. Mann R., Mulligan R. C., Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell. 1983 May;33(1):153–159. doi: 10.1016/0092-8674(83)90344-6. [DOI] [PubMed] [Google Scholar]
  18. Parker B. A., Stark G. R. Regulation of simian virus 40 transcription: sensitive analysis of the RNA species present early in infections by virus or viral DNA. J Virol. 1979 Aug;31(2):360–369. doi: 10.1128/jvi.31.2.360-369.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Peabody D. S., Berg P. Termination-reinitiation occurs in the translation of mammalian cell mRNAs. Mol Cell Biol. 1986 Jul;6(7):2695–2703. doi: 10.1128/mcb.6.7.2695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Peabody D. S., Subramani S., Berg P. Effect of upstream reading frames on translation efficiency in simian virus 40 recombinants. Mol Cell Biol. 1986 Jul;6(7):2704–2711. doi: 10.1128/mcb.6.7.2704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Reik W., Weiher H., Jaenisch R. Replication-competent Moloney murine leukemia virus carrying a bacterial suppressor tRNA gene: selective cloning of proviral and flanking host sequences. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1141–1145. doi: 10.1073/pnas.82.4.1141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rowe W. P., Pugh W. E., Hartley J. W. Plaque assay techniques for murine leukemia viruses. Virology. 1970 Dec;42(4):1136–1139. doi: 10.1016/0042-6822(70)90362-4. [DOI] [PubMed] [Google Scholar]
  23. Shinnick T. M., Lerner R. A., Sutcliffe J. G. Nucleotide sequence of Moloney murine leukaemia virus. Nature. 1981 Oct 15;293(5833):543–548. doi: 10.1038/293543a0. [DOI] [PubMed] [Google Scholar]
  24. Simonsen C. C., Levinson A. D. Isolation and expression of an altered mouse dihydrofolate reductase cDNA. Proc Natl Acad Sci U S A. 1983 May;80(9):2495–2499. doi: 10.1073/pnas.80.9.2495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Stuhlmann H., Cone R., Mulligan R. C., Jaenisch R. Introduction of a selectable gene into different animal tissue by a retrovirus recombinant vector. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7151–7155. doi: 10.1073/pnas.81.22.7151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Subramani S., Mulligan R., Berg P. Expression of the mouse dihydrofolate reductase complementary deoxyribonucleic acid in simian virus 40 vectors. Mol Cell Biol. 1981 Sep;1(9):854–864. doi: 10.1128/mcb.1.9.854. [DOI] [PMC free article] [PubMed] [Google Scholar]

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