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. 1989 Nov;63(11):4857–4865. doi: 10.1128/jvi.63.11.4857-4865.1989

Transfer of a mutant dihydrofolate reductase gene into pre- and postimplantation mouse embryos by a replication-competent retrovirus vector.

H Stuhlmann 1, R Jaenisch 1, R C Mulligan 1
PMCID: PMC251124  PMID: 2795720

Abstract

In order to explore the potential of retrovirus vectors for efficiently transferring foreign genes into mouse embryos, a replication-competent recombinant Moloney murine leukemia virus (Mo-MLV) vector carrying a mutant dihydrofolate reductase (DHFR) cDNA insert in the U3 region of the viral long terminal repeat was used to infect pre- and postimplantation embryos. When preimplantation mouse embryos were infected with the vector, as expected, the provirus integrated into the embryos and the germ line with the same efficiency as that observed with wild-type Mo-MLV, leading to inactivation of the recombinant virus. In contrast, when postimplantation mouse embryos were microinjected with virus-producing cells, between 90 to 100% of the surviving animals proved to be infected with the virus. The recombinant virus spread as efficiently as wild-type Mo-MLV in the infected embryos, resulting in up to three to five proviral copies per genome in heart, thymus, and brain tissues. Substantial expression of mutant DHFR*-coding viral message was found in all somatic tissues analyzed, the amounts correlating with the proviral copy number in the respective organ. These results suggest that replication-competent vectors are useful for efficient transfer and expression of foreign genes into tissues or whole animals when virus spread is needed.

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Selected References

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

  1. Baty D., Barrera-Saldana H. A., Everett R. D., Vigneron M., Chambon P. Mutational dissection of the 21 bp repeat region of the SV40 early promoter reveals that it contains overlapping elements of the early-early and late-early promoters. Nucleic Acids Res. 1984 Jan 25;12(2):915–932. doi: 10.1093/nar/12.2.915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Gordon J. W. A foreign dihydrofolate reductase gene in transgenic mice acts as a dominant mutation. Mol Cell Biol. 1986 Jun;6(6):2158–2167. doi: 10.1128/mcb.6.6.2158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Hunt L. A., Brown D. W., Robinson H. L., Naeve C. W., Webster R. G. Retrovirus-expressed hemagglutinin protects against lethal influenza virus infections. J Virol. 1988 Aug;62(8):3014–3019. doi: 10.1128/jvi.62.8.3014-3019.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Huszar D., Balling R., Kothary R., Magli M. C., Hozumi N., Rossant J., Bernstein A. Insertion of a bacterial gene into the mouse germ line using an infectious retrovirus vector. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8587–8591. doi: 10.1073/pnas.82.24.8587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Isola L. M., Gordon J. W. Systemic resistance to methotrexate in transgenic mice carrying a mutant dihydrofolate reductase gene. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9621–9625. doi: 10.1073/pnas.83.24.9621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jaenisch R., Harbers K., Schnieke A., Löhler J., Chumakov I., Jähner D., Grotkopp D., Hoffmann E. Germline integration of moloney murine leukemia virus at the Mov13 locus leads to recessive lethal mutation and early embryonic death. Cell. 1983 Jan;32(1):209–216. doi: 10.1016/0092-8674(83)90511-1. [DOI] [PubMed] [Google Scholar]
  10. Jaenisch R., Jähner D., Nobis P., Simon I., Löhler J., Harbers K., Grotkopp D. Chromosomal position and activation of retroviral genomes inserted into the germ line of mice. Cell. 1981 May;24(2):519–529. doi: 10.1016/0092-8674(81)90343-3. [DOI] [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. Jaenisch R., Schnieke A., Harbers K. Treatment of mice with 5-azacytidine efficiently activates silent retroviral genomes in different tissues. Proc Natl Acad Sci U S A. 1985 Mar;82(5):1451–1455. doi: 10.1073/pnas.82.5.1451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jähner D., Haase K., Mulligan R., Jaenisch R. Insertion of the bacterial gpt gene into the germ line of mice by retroviral infection. Proc Natl Acad Sci U S A. 1985 Oct;82(20):6927–6931. doi: 10.1073/pnas.82.20.6927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jähner D., Jaenisch R. Integration of Moloney leukaemia virus into the germ line of mice: correlation between site of integration and virus activation. Nature. 1980 Oct 2;287(5781):456–458. doi: 10.1038/287456a0. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Keller G., Paige C., Gilboa E., Wagner E. F. Expression of a foreign gene in myeloid and lymphoid cells derived from multipotent haematopoietic precursors. Nature. 1985 Nov 14;318(6042):149–154. doi: 10.1038/318149a0. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Lemischka I. R., Raulet D. H., Mulligan R. C. Developmental potential and dynamic behavior of hematopoietic stem cells. Cell. 1986 Jun 20;45(6):917–927. doi: 10.1016/0092-8674(86)90566-0. [DOI] [PubMed] [Google Scholar]
  19. Nobis P., Jaenisch R. Passive immunotherapy prevents expression of endogenous Moloney virus and amplification of proviral DNA in BALB/Mo mice. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3677–3681. doi: 10.1073/pnas.77.6.3677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Price J., Turner D., Cepko C. Lineage analysis in the vertebrate nervous system by retrovirus-mediated gene transfer. Proc Natl Acad Sci U S A. 1987 Jan;84(1):156–160. doi: 10.1073/pnas.84.1.156. [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. Rubenstein J. L., Nicolas J. F., Jacob F. Introduction of genes into preimplantation mouse embryos by use of a defective recombinant retrovirus. Proc Natl Acad Sci U S A. 1986 Jan;83(2):366–368. doi: 10.1073/pnas.83.2.366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sanes J. R., Rubenstein J. L., Nicolas J. F. Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos. EMBO J. 1986 Dec 1;5(12):3133–3142. doi: 10.1002/j.1460-2075.1986.tb04620.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Soriano P., Jaenisch R. Retroviruses as probes for mammalian development: allocation of cells to the somatic and germ cell lineages. Cell. 1986 Jul 4;46(1):19–29. doi: 10.1016/0092-8674(86)90856-1. [DOI] [PubMed] [Google Scholar]
  27. Stewart C. L., Schuetze S., Vanek M., Wagner E. F. Expression of retroviral vectors in transgenic mice obtained by embryo infection. EMBO J. 1987 Feb;6(2):383–388. doi: 10.1002/j.1460-2075.1987.tb04766.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Stuhlmann H., Jaenisch R., Mulligan R. C. Construction and properties of replication-competent murine retroviral vectors encoding methotrexate resistance. Mol Cell Biol. 1989 Jan;9(1):100–108. doi: 10.1128/mcb.9.1.100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Turner D. L., Cepko C. L. A common progenitor for neurons and glia persists in rat retina late in development. Nature. 1987 Jul 9;328(6126):131–136. doi: 10.1038/328131a0. [DOI] [PubMed] [Google Scholar]
  31. Williams D. A., Lemischka I. R., Nathan D. G., Mulligan R. C. Introduction of new genetic material into pluripotent haematopoietic stem cells of the mouse. Nature. 1984 Aug 9;310(5977):476–480. doi: 10.1038/310476a0. [DOI] [PubMed] [Google Scholar]
  32. Wilson J. M., Jefferson D. M., Chowdhury J. R., Novikoff P. M., Johnston D. E., Mulligan R. C. Retrovirus-mediated transduction of adult hepatocytes. Proc Natl Acad Sci U S A. 1988 May;85(9):3014–3018. doi: 10.1073/pnas.85.9.3014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wolff J. A., Yee J. K., Skelly H. F., Moores J. C., Respess J. G., Friedmann T., Leffert H. Expression of retrovirally transduced genes in primary cultures of adult rat hepatocytes. Proc Natl Acad Sci U S A. 1987 May;84(10):3344–3348. doi: 10.1073/pnas.84.10.3344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. van der Putten H., Botteri F. M., Miller A. D., Rosenfeld M. G., Fan H., Evans R. M., Verma I. M. Efficient insertion of genes into the mouse germ line via retroviral vectors. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6148–6152. doi: 10.1073/pnas.82.18.6148. [DOI] [PMC free article] [PubMed] [Google Scholar]

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