Inefficiency of expression of luciferase reporter from transfected murine leukaemia proviral DNA may be partially overcome by providing a strong polyadenylation signal Free

Abstract

HepG2 human hepatoma cells were transfected with the luciferase reporter gene, linked with a liver-specific enhancer plus a minimal promoter, contained within either pBR/pUC or Moloney murine leukaemia virus (MMLV) proviral plasmid contexts. Reporter expression from the proviral plasmid was decreased 10- to 20-fold, regardless of whether or not the orientation within the proviral DNA was appropriate for the use of the poly(A) signal in the 3′ long terminal repeat (LTR). Efficient reporter expression was restored when the proviral transcription unit was provided with a simian virus 40 poly(A) signal. These results imply that the MMLV LTR poly(A) signal is inefficient. Therefore, strategies to maximize expression of internal transcription units from retroviral vectors should include the provision of an efficient (unidirectional) poly(A) signal (its requiring insertion in the reverse orientation to that of viral transcription).

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1991-07-01
2024-03-29
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References

  1. Bender M. A., Palmer T. D., Gelinas R. E., Miller A. D. 1987; Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region. Journal of Virology 61:1639–1646
    [Google Scholar]
  2. Buchman A. R., Burnett L., Berg P. 1981; The SV40 nucleotide sequence. Appendix A. In DNA Tumor Viruses, 2nd. edn. pp. 799–841 Edited by Tooze J. New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  3. Cone R. D., Reilly E. B., Eisen H. N., Mulligan R. C. 1987; Tissue-specific expression of functionally rearranged lambda-1 Ig gene through a retrovirus vector. Science 236:954–957
    [Google Scholar]
  4. De Simone V., Ciliberto G., Hardon E., Paonessa G., Palla F., Lundberg L., Cortese R. 1987; Cis- and trans-acting elements responsible for the cell-specific expression of the human alpha-1-antitrypsin gene. EMBO Journal 6:2759–2766
    [Google Scholar]
  5. De Wet J. R., Wood K. V., Deluca M., Helinski D. R., Subramani S. 1987; Firefly luciferase gene: structure and expression in mammalian cells. Molecular and Cellular Biology 7:725–737
    [Google Scholar]
  6. Iwasaki K., Temin H. M. 1990; The efficiency of RNA 3′-end formation is determined by the distance between the cap site and the poly(A) site in spleen necrosis virus. Genes and Development 4:2299–2307
    [Google Scholar]
  7. Kadesch T., Berg P. 1986; Effects of the position of the simian virus 40 enhancer on expression of multiple transcription units in a single plasmid. Molecular and Cellular Biology 6:2593–2601
    [Google Scholar]
  8. Maxwell I. H., Maxwell F. 1988; Electroporation of mammalian cells with a firefly luciferase expression plasmid: kinetics of transient expression differ markedly among cell types. DNA 7:557–562
    [Google Scholar]
  9. Maxwell I. H., Harrison G. S., Wood W. M., Maxwell F. 1989; A DNA cassette containing a trimerized SV40 polyadenylation signal which efficiently blocks spurious plasmid-initiated transcription. Bio/Techniques 7:276–280
    [Google Scholar]
  10. Maxwell I. H., Maxwell F., Glode L. M. 1986; Regulated expression of a diphtheria toxin A-chain gene transfected into human cells: possible strategy for inducing cancer cell suicide. Cancer Research 46:4660–4664
    [Google Scholar]
  11. Miller A. D., Rosman G. L. 1989; Improved retroviral vectors for gene transfer and expression. Bio/Techniques 7:980–988
    [Google Scholar]
  12. Nordeen S. K. 1988; Luciferase reporter gene vectors for analysis of promoters and enhancers. Bio/Techniques 6:454–457
    [Google Scholar]
  13. Swain A., Coffin J. M. 1989; Polyadenylation at correct sites in genome RNA is not required for retrovirus replication or genome encapsidation. Journal of Virology 63:3301–3306
    [Google Scholar]
  14. Temin H. M. 1986; Retrovirus vectors for gene transfer: efficient integration into and expression of exogenous DNA in vertebrate cell genomes. In Gene Transfer pp. 149–187 Edited by Kucherlapati R. New York & London: Plenum Press;
    [Google Scholar]
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