Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1985 Apr;82(8):2422–2426. doi: 10.1073/pnas.82.8.2422

Isolation of embryonal carcinoma cell lines that express integrated recombinant genes flanked by the Moloney murine leukemia virus long terminal repeat.

M Taketo, E Gilboa, M I Sherman
PMCID: PMC397570  PMID: 3857593

Abstract

We have used a producer NIH 3T3 cell line that secretes, together with the helper Moloney murine leukemia virus (Mo-MuLV), a transducing recombinant virus containing the neomycin-resistance gene linked to the Mo-MuLV long terminal repeat (LTR). By infecting three embryonal carcinoma cell lines, PCC4.aza1R, F9tk-, and Nulli-SCC1, with this recombinant virus, we have isolated many transductant clones that stably express the integrated neomycin-resistance gene. These clonal transductant lines consist of undifferentiated embryonal carcinoma cells as judged by morphology, tumorigenicity in 129/Sv mice, and cell-surface antigenic markers. Analysis of the integrated recombinant viral genes by Southern blot hybridization revealed that some of the lines have single copies, whereas others have multiple copies, probably in multiple sites. Although these transductant lines contained many copies of helper Mo-MuLV integrated in the cellular genome, expression of these helper viruses was not detected either by reverse transcriptase activity or by X-C plaque assay. Two F9tk--derived, G418-resistant transductant lines were superinfected with a second recombinant transducing virus that contains the herpes simplex virus thymidine kinase gene flanked by the Mo-MuLV LTR. The frequency of transduction to yield clones able to grow in hypoxanthine/aminopterin/thymidine medium was similar to that of the parental F9tk- cells. These results suggest that the expression of the neomycin-resistance gene, linked to MoMuLV LTR in the transductant embryonal carcinoma cell clones, is due to a cisacting mechanism(s).

Full text

PDF
2422

Images in this article

2424Image
on p.2424
2425Image
on p.2425

Selected References

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

  1. Blin N., Stafford D. W. A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res. 1976 Sep;3(9):2303–2308. doi: 10.1093/nar/3.9.2303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boccara M., Kelly F. Expression of polyoma virus in heterokaryons between embryonal carcinoma cells and differentiated cells. Virology. 1978 Oct 1;90(1):147–150. doi: 10.1016/0042-6822(78)90342-2. [DOI] [PubMed] [Google Scholar]
  3. Chattopadhyay S. K., Lowy D. R., Teich N. M., Levine A. S., Rowe W. P. Qualitative and quantitative studies of AKR-type murine leukemia virus sequences in mouse DNA. Cold Spring Harb Symp Quant Biol. 1975;39(Pt 2):1085–1101. doi: 10.1101/sqb.1974.039.01.124. [DOI] [PubMed] [Google Scholar]
  4. Fujimura F. K., Deininger P. L., Friedmann T., Linney E. Mutation near the polyoma DNA replication origin permits productive infection of F9 embryonal carcinoma cells. Cell. 1981 Mar;23(3):809–814. doi: 10.1016/0092-8674(81)90445-1. [DOI] [PubMed] [Google Scholar]
  5. Gautsch J. W. Embryonal carcinoma stem cells lack a function required for virus replication. Nature. 1980 May 8;285(5760):110–112. doi: 10.1038/285110a0. [DOI] [PubMed] [Google Scholar]
  6. Gautsch J. W., Wilson M. C. Delayed de novo methylation in teratocarcinoma suggests additional tissue-specific mechanisms for controlling gene expression. Nature. 1983 Jan 6;301(5895):32–37. doi: 10.1038/301032a0. [DOI] [PubMed] [Google Scholar]
  7. Gilboa E., Kolbe M., Noonan K., Kucherlapati R. Construction of a mammalian transducing vector from the genome of Moloney murine leukemia virus. J Virol. 1982 Dec;44(3):845–851. doi: 10.1128/jvi.44.3.845-851.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gmür R., Solter D., Knowles B. B. Independent regulation of H-2K and H-2D gene expression in murine teratocarcinoma somatic cell hybrids. J Exp Med. 1980 Jun 1;151(6):1349–1359. doi: 10.1084/jem.151.6.1349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hartley J. W., Rowe W. P. Naturally occurring murine leukemia viruses in wild mice: characterization of a new "amphotropic" class. J Virol. 1976 Jul;19(1):19–25. doi: 10.1128/jvi.19.1.19-25.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jainchill J. L., Aaronson S. A., Todaro G. J. Murine sarcoma and leukemia viruses: assay using clonal lines of contact-inhibited mouse cells. J Virol. 1969 Nov;4(5):549–553. doi: 10.1128/jvi.4.5.549-553.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jetten A. M., Jetten M. E., Sherman M. I. Stimulation of differentiation of several murine embryonal carcinoma cell lines by retinoic acid. Exp Cell Res. 1979 Dec;124(2):381–391. doi: 10.1016/0014-4827(79)90213-1. [DOI] [PubMed] [Google Scholar]
  12. Jorgensen R. A., Rothstein S. J., Reznikoff W. S. A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Mol Gen Genet. 1979;177(1):65–72. doi: 10.1007/BF00267254. [DOI] [PubMed] [Google Scholar]
  13. Katinka M., Yaniv M., Vasseur M., Blangy D. Expression of polyoma early functions in mouse embryonal carcinoma cells depends on sequence rearrangements in the beginning of the late region. Cell. 1980 Jun;20(2):393–399. doi: 10.1016/0092-8674(80)90625-x. [DOI] [PubMed] [Google Scholar]
  14. Linney E., Davis B., Overhauser J., Chao E., Fan H. Non-function of a Moloney murine leukaemia virus regulatory sequence in F9 embryonal carcinoma cells. 1984 Mar 29-Apr 4Nature. 308(5958):470–472. doi: 10.1038/308470a0. [DOI] [PubMed] [Google Scholar]
  15. Littlefield J. W., Felix J. S. Rescue of terminally differentiating teratocarcinoma cells by fusion to undifferentiated parental cells. Somatic Cell Genet. 1982 Nov;8(6):743–757. doi: 10.1007/BF01543016. [DOI] [PubMed] [Google Scholar]
  16. McCue P. A., Matthaei K. I., Taketo M., Sherman M. I. Differentiation-defective mutants of mouse embryonal carcinoma cells: response to hexamethylenebisacetamide and retinoic acid. Dev Biol. 1983 Apr;96(2):416–426. doi: 10.1016/0012-1606(83)90179-3. [DOI] [PubMed] [Google Scholar]
  17. Niwa O., Yokota Y., Ishida H., Sugahara T. Independent mechanisms involved in suppression of the Moloney leukemia virus genome during differentiation of murine teratocarcinoma cells. Cell. 1983 Apr;32(4):1105–1113. doi: 10.1016/0092-8674(83)90294-5. [DOI] [PubMed] [Google Scholar]
  18. Ross J., Scolnick E. M., Todaro G. J., Aaronson S. A. Separation of murine cellular and murine leukaemia virus DNA polymerases. Nat New Biol. 1971 Jun 9;231(23):163–167. doi: 10.1038/newbio231163a0. [DOI] [PubMed] [Google Scholar]
  19. Rowe W. P., Hartley J. W., Lander M. R., Pugh W. E., Teich N. Noninfectious AKR mouse embryo cell lines in which each cell has the capacity to be activated to produce infectious murine leukemia virus. Virology. 1971 Dec;46(3):866–876. doi: 10.1016/0042-6822(71)90087-0. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Sherman M. I., Miller R. A. F9 embryonal carcinoma cells can differentiate into endoderm-like cells. Dev Biol. 1978 Mar;63(1):27–34. doi: 10.1016/0012-1606(78)90110-0. [DOI] [PubMed] [Google Scholar]
  22. Solter D., Knowles B. B. Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA-1). Proc Natl Acad Sci U S A. 1978 Nov;75(11):5565–5569. doi: 10.1073/pnas.75.11.5565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sorge J., Cutting A. E., Erdman V. D., Gautsch J. W. Integration-specific retrovirus expression in embryonal carcinoma cells. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6627–6631. doi: 10.1073/pnas.81.21.6627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  25. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  26. Stewart C. L., Stuhlmann H., Jähner D., Jaenisch R. De novo methylation, expression, and infectivity of retroviral genomes introduced into embryonal carcinoma cells. Proc Natl Acad Sci U S A. 1982 Jul;79(13):4098–4102. doi: 10.1073/pnas.79.13.4098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Strickland S. Mouse teratocarcinoma cells: prospects for the study of embryogenesis and neoplasia. Cell. 1981 May;24(2):277–278. doi: 10.1016/0092-8674(81)90313-5. [DOI] [PubMed] [Google Scholar]
  28. Swartzendruber D. E., Friedrich T. D., Lehman J. M. Resistance of teratocarcinoma stem cells to infection with simian virus 40: early events. J Cell Physiol. 1977 Oct;93(1):25–30. doi: 10.1002/jcp.1040930105. [DOI] [PubMed] [Google Scholar]
  29. Swartzendruber D. E., Lehman J. M. Neoplastic differentiation: interaction of simian virus 40 and polyoma virus with murine teratocarcinoma cells in vitro. J Cell Physiol. 1975 Apr;85(2 Pt 1):179–187. doi: 10.1002/jcp.1040850204. [DOI] [PubMed] [Google Scholar]
  30. Teich N. M., Weiss R. A., Martin G. R., Lowy D. R. Virus infection of murine teratocarcinoma stem cell lines. Cell. 1977 Dec;12(4):973–982. doi: 10.1016/0092-8674(77)90162-3. [DOI] [PubMed] [Google Scholar]
  31. Wei C. M., Gibson M., Spear P. G., Scolnick E. M. Construction and isolation of a transmissible retrovirus containing the src gene of Harvey murine sarcoma virus and the thymidine kinase gene of herpes simplex virus type 1. J Virol. 1981 Sep;39(3):935–944. doi: 10.1128/jvi.39.3.935-944.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wigler M., Silverstein S., Lee L. S., Pellicer A., Cheng Y. c., Axel R. Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells. Cell. 1977 May;11(1):223–232. doi: 10.1016/0092-8674(77)90333-6. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES