Advertisement

Cytotechnology

, 24:31 | Cite as

Effects of an internal transcription unit and its orientation on retrovirus titre and expression

  • Simon J.H. Hettle
  • Chris Darnbrough
  • Patricia L. Watts
  • Caroline MacDonald
Article
  • 30 Downloads

Abstract

Using the retroviral vector pMsp, constructs were produced with different coding sequences under the control of the Herpes simplex virus type 1 (HSV) thymidine kinase (tk) promoter, with the internal coding sequence in the same or reverse orientations with respect to long terminal repeat (LTR)-driven transcription and with or without an internal tk polyadenylation (polyA) signal. Following introduction of these constructs into ecotropic or amphotropic packaging cell lines by transfection or infection, it was found that, most consistently, those constructs in which the internal coding sequence/polyA signal component was inserted in the same orientation as LTR-driven transcription produced lower titres of virus than those in which this component was inserted in the reverse orientation. Also, in a construct containing a coding sequence in the same orientation as LTR-driven transcription, but lacking an internal polyA signal, virus titre was much greater than in the corresponding construct possessing the internal polyA signal. Additionally, although functional assays have previously demonstrated expression of the inserted sequences, transcription from the internal tk promoter was inefficient in all these constructs.

insulin-like growth factors myc gene polyadenylation signal retroviral vectors tk promoter 

References

  1. Artelt P, Grannemann R, Stocking C, Friel J, Bartsch J & Hauser H (1991) The prokaryotic neomycin-resistance-encoding gene acts as a transcriptional silencer in eukaryotic cells. Gene 99: 249–254.PubMedCrossRefGoogle Scholar
  2. Bell GI, Merryweather JP, Sanchez-Pescador R, Stempien MM, Priestley L, Scott J & Rall LB (1984) Sequence of a cDNA clone encoding human preproinsulin-like growth factor II. Nature 310: 775–777.PubMedCrossRefGoogle Scholar
  3. Brown AMC & Scott MRD (1987) Retroviral vectors, in Glover DM (ed.) DNA Cloning -- A Practical Approach. Vol. 3 (pp. 189–212) IRL Press, Oxford.Google Scholar
  4. Cepko CL, Roberts BE & Mulligan RC (1984) Construction and application of a highly transmissible murine retrovirus shuttle vector. Cell 37: 1053–1062.PubMedCrossRefGoogle Scholar
  5. Chomczynski P & Sacchi N (1987) Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156–159.PubMedCrossRefGoogle Scholar
  6. Danos O & Mulligan RC (1988) Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges. Proc. Natl. Acad. Sci. USA. 85: 6460–6464.PubMedCrossRefGoogle Scholar
  7. Darnbrough C & MacDonald C (1991) Enhanced growth characteristics of hybridoma cells infected with myc-and ras-containing retroviruses. Exp. Cell Res. 195: 263–268.PubMedCrossRefGoogle Scholar
  8. Darnbrough C, Slater S, Vass MAV & MacDonald C (1992) Immortalization of murine primary spleen cells by v-myc, v-ras and v-raf. Exp. Cell Res. 201: 273–283.PubMedCrossRefGoogle Scholar
  9. Emerman M & Temin HM (1984) Genes with promoters in retrovirus vectors can be independently suppressed by an epigenetic mechanism. Cell 39: 459–467.CrossRefGoogle Scholar
  10. Emerman M & Temin HM (1986) Quantitative analysis of gene suppression in integrated retrovirus vectors. Mol. Cell. Biol. 6: 792–800.PubMedGoogle Scholar
  11. Gorman C (1985) High efficiency gene transfer into mammalian cells, in Glover DM (ed.) DNA Cloning -- A Practical Approach. Vol. 2 (pp. 143–190) IRL Press, Oxford.Google Scholar
  12. Green PJ, Pines O & Inouye M (1986) The role of antisense RNA in gene regulation. Ann. Rev. Biochem. 55: 569–597.PubMedCrossRefGoogle Scholar
  13. Jansen M, van Schaik FMA, Ricker AT, Bullock B, Woods DE, Gabbay KH, Nussbaum AL, Sussenbach JS & Van den Brande JL (1983) Sequence of cDNA encoding human insulin-like growth factor I precursor. Nature 306: 609–611.PubMedCrossRefGoogle Scholar
  14. Kreuzburg-Duffy U & MacDonald C (1991) Establishment of immortalized cell lines from mouse peritoneal macrophages following transformation with SV40 early region DNA deleted at the origin of replication. Immunology 72: 368–372.PubMedGoogle Scholar
  15. Mann R, Mulligan RC & Baltimore DB (1983) Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell 33: 153–159.PubMedCrossRefGoogle Scholar
  16. Miller AD & Buttimore C (1986) Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol. Cell. Biol. 6: 2895–2902.PubMedGoogle Scholar
  17. Miller AD, Law MF & Verma IM (1985) Generation of helper-free amphotropic retroviruses that transduce a dominant acting methotrexate-resistant dihydrofolate reductase gene. Mol. Cell. Biol. 5: 431–437.PubMedGoogle Scholar
  18. Miller AD, Trauber DR & Buttimore C (1986) Factors involved in production of helper virus free retrovirus vectors. Somatic Cell Mol. Genet. 12: 175–183.PubMedCrossRefGoogle Scholar
  19. Miyao Y, Shimizu K, Tamura M, Yamada M, Tamura K, Nakahira K, Kuriyama S, Hayakawa T & Ikenaka K (1995) A simplified general method for determination of recombinant retrovirus titres. Cell Struct. Func. 20: 177–183.CrossRefGoogle Scholar
  20. Ostertag W, Seliger B, Kollek R, Stocking C, Bergholz U & Smadja-Joffe F (1986) The myeloproliferative sarcoma virus retains transforming functions after introduction of a dominant selectable marker gene. J. Gen. Virol. 67: 1361–1371.PubMedCrossRefGoogle Scholar
  21. Rowe WP, Pugh WE & Hartley JW (1970) Plaque assay techniques for murine leukaemia viruses. Virology 42: 1136–1139.PubMedCrossRefGoogle Scholar
  22. Shimotohono K & Temin HM (1981) Formation of infectious progeny virus after insertion of herpes simplex thymidine kinase gene into DNA of an avian retrovirus. Cell 26: 67–77.CrossRefGoogle Scholar
  23. Stanton LW, Fahrlander PD, Tesser PM & Marcu KB (1984) Nucleotide sequence comparisons of normal and translocated murine c-myc genes. Nature 310: 423–425.PubMedCrossRefGoogle Scholar
  24. Stewart MA, Forrest D, McFarlane R, Onions D, Wilkie N & Neil J C (1986) conservation of the c-myc coding sequence in transduced feline v-myc genes. Virology 154: 121–134.PubMedCrossRefGoogle Scholar
  25. Watts PL (1990) Retroviral vector-mediated expression of cloned growth factor genes in mammalian cells. Ph.D. thesis, University of Strathclyde.Google Scholar
  26. Watts PL & MacDonald C (1990) Production of biologically active insulin-like growth factor by cell transfected with a recombinant retroviral vector encodiNG IGF-I, in Spier R, Griffiths JB & Meignier B (eds.) Production of biologicals from animal cells in culture. (pp. 726–731) Butterworth-Heinemann, Oxford.Google Scholar
  27. Yee J-K, Moores JC, Jolly DJ, Wolff JA, Respess JG & Friedmann T (1987) Gene expression from transcriptionally disabled retroviral vectors. Proc. Natl. Acad. Sci. USA. 84: 5197–5201.PubMedCrossRefGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1997

Authors and Affiliations

  • Simon J.H. Hettle
    • 1
  • Chris Darnbrough
    • 2
  • Patricia L. Watts
    • 2
  • Caroline MacDonald
    • 1
  1. 1.Department of Biological SciencesUniversity of Paisley, PaisleyScotlandUK
  2. 2.Department of ImmunologyUniversity of StrathclydeScotlandUK

Personalised recommendations