Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1983 Dec;3(12):2241–2249. doi: 10.1128/mcb.3.12.2241

Construction of a helper cell line for avian reticuloendotheliosis virus cloning vectors.

S Watanabe, H M Temin
PMCID: PMC370095  PMID: 6318091

Abstract

We wished to construct cell lines that supply the gene products of gag, pol, and env for the growth of replication-defective reticuloendotheliosis retrovirus vectors without production of the helper virus. To do this, first we located by S1 mapping the donor and acceptor splice sites of reticuloendotheliosis virus strain A. The donor splice site is ca. 850 base pairs from the 5' end of proviral DNA. It is close to or overlaps the encapsidation sequences for viral RNA. The splice acceptor site is ca. 5.6 kilobase pairs from the 5' end of proviral DNA. Therefore, the encapsidation sequences and the donor splice site were removed from viral DNA to give expression of the gag and pol genes without virus production. The promoter in the long terminal repeat was fused to a site near the first ATG codon of the env gene, thereby deleting the encapsidation sequences and the gag and pol genes to give expression of the env gene without virus production. The permissive canine cell line D17 was transfected with the two modified viral DNAs. Two cell clones that contain both modified viral DNAs support the production of replication-defective spleen necrosis virus-thymidine kinase recombinant retrovirus vectors without the production of helper virus. To prevent recombination, the vector contains deletions that overlap with deletions in the integrated helper virus DNAs. This helper cell-vector system will be useful to derive infectious recombinant virus stocks of high titer (over 10(5) thymidine kinase transforming units per ml) which are able to infect avian, rat, and dog cells without the aid of helper virus.

Full text

PDF
2241

Images in this article

2244Image
on p.2244
2244Image
on p.2244
2245Image
on p.2245
2245Image
on p.2245
2247Image
on p.2247

Selected References

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

  1. Baker B., Robison H., Varmus H. E., Bishop J. M. Analysis of endogenous avian retrovirus DNA and RNA: viral and cellular determinants of retrovirus gene expression. Virology. 1981 Oct 15;114(1):8–22. doi: 10.1016/0042-6822(81)90248-8. [DOI] [PubMed] [Google Scholar]
  2. Benjamin T. L. Host range mutants of polyoma virus. Proc Natl Acad Sci U S A. 1970 Sep;67(1):394–399. doi: 10.1073/pnas.67.1.394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berkner K. L., Folk W. R. Polynucleotide kinase exchange reaction: quantitave assay for restriction endonuclease-generated 5'-phosphoroyl termini in DNA. J Biol Chem. 1977 May 25;252(10):3176–3184. [PubMed] [Google Scholar]
  4. Casey J., Davidson N. Rates of formation and thermal stabilities of RNA:DNA and DNA:DNA duplexes at high concentrations of formamide. Nucleic Acids Res. 1977;4(5):1539–1552. doi: 10.1093/nar/4.5.1539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen I. S., Mak T. W., O'Rear J. J., Temin H. M. Characterization of reticuloendotheliosis virus strain T DNA and isolation of a novel variant of reticuloendotheliosis virus strain T by molecular cloning. J Virol. 1981 Dec;40(3):800–811. doi: 10.1128/jvi.40.3.800-811.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Denhardt D. T. A membrane-filter technique for the detection of complementary DNA. Biochem Biophys Res Commun. 1966 Jun 13;23(5):641–646. doi: 10.1016/0006-291x(66)90447-5. [DOI] [PubMed] [Google Scholar]
  7. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  8. Hackett P. B., Swanstrom R., Varmus H. E., Bishop J. M. The leader sequence of the subgenomic mRNA's of Rous sarcoma virus is approximately 390 nucleotides. J Virol. 1982 Feb;41(2):527–534. doi: 10.1128/jvi.41.2.527-534.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Haseltine W. A., Maxam A. M., Gilbert W. Rous sarcoma virus genome is terminally redundant: the 5' sequence. Proc Natl Acad Sci U S A. 1977 Mar;74(3):989–993. doi: 10.1073/pnas.74.3.989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Katz L., Kingsbury D. T., Helinski D. R. Stimulation by cyclic adenosine monophosphate of plasmid deoxyribonucleic acid replication and catabolite repression of the plasmid deoxyribonucleic acid-protein relaxation complex. J Bacteriol. 1973 May;114(2):577–591. doi: 10.1128/jb.114.2.577-591.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Linial M. Transfer of defective avian tumor virus genomes by a Rous sarcoma virus RNA packaging mutant. J Virol. 1981 Apr;38(1):380–382. doi: 10.1128/jvi.38.1.380-382.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Mitra S. W., Chow M., Champoux J., Baltimore D. Synthesis of murine leukemia virus plus strong stop DNA initiates at a unique site. J Biol Chem. 1982 Jun 10;257(11):5983–5986. [PubMed] [Google Scholar]
  15. Mulligan R. C., Berg P. Selection for animal cells that express the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2072–2076. doi: 10.1073/pnas.78.4.2072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  17. Ross J. A precursor of globin messenger RNA. J Mol Biol. 1976 Sep 15;106(2):403–420. doi: 10.1016/0022-2836(76)90093-0. [DOI] [PubMed] [Google Scholar]
  18. SZYBALSKA E. H., SZYBALSKI W. Genetics of human cess line. IV. DNA-mediated heritable transformation of a biochemical trait. Proc Natl Acad Sci U S A. 1962 Dec 15;48:2026–2034. doi: 10.1073/pnas.48.12.2026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Sharp P. A. Speculations on RNA splicing. Cell. 1981 Mar;23(3):643–646. doi: 10.1016/0092-8674(81)90425-6. [DOI] [PubMed] [Google Scholar]
  21. Shimotohno K., Temin H. M. Formation of infectious progeny virus after insertion of herpes simplex thymidine kinase gene into DNA of an avian retrovirus. Cell. 1981 Oct;26(1 Pt 1):67–77. doi: 10.1016/0092-8674(81)90034-9. [DOI] [PubMed] [Google Scholar]
  22. Shimotohno K., Temin H. M. Loss of intervening sequences in genomic mouse alpha-globin DNA inserted in an infectious retrovirus vector. Nature. 1982 Sep 16;299(5880):265–268. doi: 10.1038/299265a0. [DOI] [PubMed] [Google Scholar]
  23. Sorge J., Hughes S. H. Polypurine tract adjacent to the U3 region of the Rous sarcoma virus genome provides a cis-acting function. J Virol. 1982 Aug;43(2):482–488. doi: 10.1128/jvi.43.2.482-488.1982. [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. Taylor J. M., Illmensee R. Site on the RNA of an avian sarcoma virus at which primer is bound. J Virol. 1975 Sep;16(3):553–558. doi: 10.1128/jvi.16.3.553-558.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Watanabe S., Temin H. M. Encapsidation sequences for spleen necrosis virus, an avian retrovirus, are between the 5' long terminal repeat and the start of the gag gene. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5986–5990. doi: 10.1073/pnas.79.19.5986. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES