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A novel T7 RNA polymerase autogene for efficient cytoplasmic expression of target genes

Gene Therapy volume 6, pages 263–270 (1999)Cite this article

Abstract

Inefficient nuclear transport of plasmid DNA continues to be a problem in nonviral vector-mediated gene transfer. This has made the cytoplasmic expression system an increasingly attractive idea. We have developed a new T7 RNA polymerase autogene for cytoplasmic expression containing both a CMV and a T7 promoter. The pCMV/T7-T7pol autogene does not encounter the problems associated with previously used autogenes. For instance, pCMV/T7-T7pol is easily amplified and purified from bacteria. Furthermore, the CMV promoter is used to drive the first round of synthesis of T7 RNA polymerase, thus negating the use of purified enzyme in the transfection complex. The endogenous T7 RNA polymerase produced from the CMV promoter could then act on the T7 promoter of pCMV/T7-T7pol in an autoregulatory mechanism. pCMV/T7-T7pol induces higher, more sustained levels (>7 days) of reporter gene expression than that observed with the previously used autogene pT7 AUTO 2C− or with the nuclear expression system pCMV-CAT. This seems to be due to the high levels of T7 RNA polymerase protein that are detected in cells transfected with pCMV/T7-T7pol. This vector also functions as an efficient autogene since at least 50 times more mRNA is transcribed from the cytoplasmic T7 promoter as compared with the nuclear CMV promoter in pCMV/T7-T7pol. Therefore, pCMV/T7-T7pol could replace existing autogenes for regeneration of T7 RNA polymerase and efficient target gene expression.

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References

  1. Capecchi MR . High efficiency transformation by direct microinjection of DNA into cultured mammalian cells Cell 1980 22: 479–488

    Article  CAS  Google Scholar 

  2. Zabner J et al. Cellular and molecular barriers to gene transfer by a cationic lipid J Biol Chem 1995 270: 18997–19007

    Article  CAS  Google Scholar 

  3. Haensler J, Szoka FC . Polyamidoamine cascade polymers mediate efficient transfection of cells in culture Bioconjugate Chem 1993 4: 372–379

    Article  CAS  Google Scholar 

  4. Gao X, Huang L . Cytoplasmic expression of a reporter gene by co-delivery of T7 RNA polymerase and T7 promoter sequence with cationic liposomes Nucleic Acids Res 1993 21: 2867–2872

    Article  CAS  Google Scholar 

  5. Dunn JJ et al. Targeting bacteriophage T7 RNA polymerase to the mammalian cell nucleus Gene 1988 68: 259–266

    Article  CAS  Google Scholar 

  6. Elroy-stein O, Moss B . Cytoplasmic expression system based on constitutive synthesis of bacteriophage T7 RNA polymerase in mammalian cells Proc Natl Acad Sci USA 1990 87: 6743–6747

    Article  CAS  Google Scholar 

  7. Chen X et al. A novel nonviral cytoplasmic gene expression system and its implications in cancer gene therapy Cancer Gene Ther 1995 2: 281–289

    CAS  PubMed  Google Scholar 

  8. Deng H, Wolff JA . Self-amplifying expression from the T7 promoter in 3T3 mouse fibroblasts Gene 1994 143: 245–249

    Article  CAS  Google Scholar 

  9. Dubendorff JW, Studier FW . Creation of a T7 autogene. Cloning and expression of the gene for bacteriophage T7 RNA polymerase under control of its cognate promoter J Mol Biol 1991 219: 61–68

    Article  CAS  Google Scholar 

  10. Chen X et al. Cancer gene therapy by direct tumor injections of a nonviral T7 vector encoding a thymidine kinase gene Hum Gene Ther 1998 9: 729–736

    Article  CAS  Google Scholar 

  11. Gao X, Jaffurs D, Robbins PD, Huang L . A sustained, cytoplasmic transgene expression system delivered by cationic liposomes Biochem Biophys Res Commun 1994 200: 1201–1206

    Article  CAS  Google Scholar 

  12. Studier FW . Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system J Mol Biol 1991 219: 37–44

    Article  CAS  Google Scholar 

  13. Elroy-stein O, Fuerst TR, Moss B . Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5′ sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system Proc Natl Acad Sci USA 1989 86: 6126–6130

    Article  CAS  Google Scholar 

  14. Gao X, Huang L . A novel cationic liposome reagent for efficient transfection of mammalian cells Biochem Biophys Res Commun 1991 179: 280–285

    Article  CAS  Google Scholar 

  15. Sorgi F, Huang L . Large scale production of DC-chol liposomes by microfluidization Int J Pharm 1996 144: 131–139

    Article  CAS  Google Scholar 

  16. Farhood H, Serbina N, Huang L . The role of dioleoyl phosphatidylethanolamine in cationic liposome mediated gene transfer Biochim Biophy Acta 1995 1235: 289–295

    Article  Google Scholar 

  17. Sambrook J, Fritsch EF, Maniatis T . Purification of closed circular DNA by equilibrium centrifugation in CsCl-Ethidium Bromide Gradients. In: Ford N, Nolan C, Ferguson M (eds) . Molecular Cloning: A Laboratory Manual, 2nd edn Cold Spring Harbor Laboratory Press: New York 1989 1.40–1.47

    Google Scholar 

  18. Sankaran L . A simple quantitative assay for chloramphenicol acetyltransferase by direct extraction of the labeled product into scintillation cocktail Anal Biochem 1992 200: 180–186

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Pharmacology, Laboratory of Drug Targeting, University of Pittsburgh School of Medicine, Pittsburgh, USA

    M Brisson, Y He, S Li, J P Yang & L Huang

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  1. M Brisson
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  2. Y He
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  3. S Li
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  4. J P Yang
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  5. L Huang
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Brisson, M., He, Y., Li, S. et al. A novel T7 RNA polymerase autogene for efficient cytoplasmic expression of target genes. Gene Ther 6, 263–270 (1999). https://doi.org/10.1038/sj.gt.3300827

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