Plat-E: an efficient and stable system for transient packaging of retroviruses (original) (raw)

Gene Therapy volume 7, pages 1063–1066 (2000)Cite this article

Abstract

A potent retrovirus packaging cell line named Platinum-E (Plat-E) was generated based on the 293T cell line. Plat-E is superior to existing packaging cell lines regarding efficiency, stability and safety. The novel packaging constructs utilized in establishment of Plat-E ensure high and stable expression of viral structural proteins. Conventional packaging constructs made use of the promoter of MuLV-LTR for expression of viral structural genes gag-pol and env, while our packaging constructs utilized the EF1α promoter, which is 100-fold more potent than the MuLV-LTR in 293T cells in combination with the Kozak's consensus sequence upstream of the initiation codon resulting in high expression of virus structural proteins in Plat-E cells. To maintain the high titers of retroviruses under drug selection pressure, we inserted the IRES (internal ribosome entry site) sequence between the gene encoding gag-pol or env, and the gene encoding a selectable marker in the packaging constructs. Plat-E cells can stably produce retroviruses with an average titer of 1 × 10_7_/ml for at least 4 months. In addition, as we used only the coding sequences of viral structural genes to avoid inclusion of unnecessary retrovirus sequences in the packaging constructs, the probability of generating the replication competent retroviruses (RCR) by recombination can virtually be ruled out.

This is a preview of subscription content, access via your institution

Access options

Subscribe to this journal

Receive 6 print issues and online access

$259.00 per year

only $43.17 per issue

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Additional access options:

Similar content being viewed by others

References

  1. Miller AD, Buttimore C . Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production Mol Cell Biol 1986 6: 2983–2902
    Article Google Scholar
  2. Pear WS, Nolan GP, Scott ML, Baltimore D . Production of high-titer helper-free retroviruses by transient transfection Proc Natl Acad Sci USA 1993 90: 8392–8396
    Article CAS Google Scholar
  3. Onishi M et al. Application of retrovirus-mediated expression cloning Exp Hematol 1996 24: 324–329
    CAS PubMed Google Scholar
  4. http://www.stanford.edu/group/nolan/
  5. Gattas IR, Sanesm JR, Major JE . The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in cultured cells and in embryo Mol Cell Biol 1991 11: 5848–5859
    Article Google Scholar
  6. Fiering SN et al. Improved FACS-Gal: flow cytometric analysis and sorting of viable eukaryotic cells expressing reporter gene constructs Cytometry 1991 12: 291–301
    Article CAS Google Scholar
  7. Dubridge RB, Tang P, Hsia HC . Analysis of mutation in human cells by using an Epstein–Barr virus shuttle system Mol Cell Biol 1987 7: 379–387
    Article CAS Google Scholar
  8. Nosaka T et al. STAT5 as a molecular regulator of proliferation, differentiation, and apoptosis in hematopoietic cells EMBO J 1999 18: 4754–4765
    Article CAS Google Scholar
  9. Mizushima S, Nagata S . pEF-BOS, a powerful mammalian expression vector Nucleic Acids Res 1990 18: 5332
    Article Google Scholar
  10. Kitamura T et al. Efficient screening of retroviral cDNA expression libraries Proc Natl Acad Sci USA 1995 92: 9146–9150
    Article CAS Google Scholar
  11. Mathias S et al. Reverse transcriptase encoded by a human transposable element Science 1991 254: 1808–1810
    Article CAS Google Scholar
  12. Evans LH et al. A neutralizable epitope common to the envelope glycoproteins of ecotropic, polytropic, xenotropic, and amphotropic murine leukeia viruses J Virol 1990 64: 6176–6183
    CAS PubMed PubMed Central Google Scholar
  13. Rowe WP, Pugh WE, Hartly JW . Plaque assay techniques for murine leukemia viruses Virology 1970 42: 1136–1139
    Article CAS Google Scholar
  14. Yoshimatsu T et al. Improvement of retroviral packaging cell lines by introducing the polyomavirus early region Hum Gene Ther 1998 9: 161–172
    Article CAS Google Scholar

Download references

Acknowledgements

We would like to thank Dr Hiroo Hoshino (Department of Hygiene and Virology, Gunma University School of Medicine) for his kind gift of MoMuLV-infected C3H2K cells, and Dr Leonard H Evans (Laboratory of Persistent Viral Disease, National Institute of Allergy and Infectious Disease) for anti-Env antibody, and Dr Kunitada Shimotohno (Department of Viral Oncology, Institute for Virus Research, Kyoto University) for his valuable discussions. We also thank Ms Mariko Ohara for her providing language assistance. This work was supported in part by grant-in-aid from the Ministry of Education, Science, Sports, and Culture and the Ministry of Health and Welfare of Japan. The Department of Hematopoietic Factors is supported by Chugai Pharmaceutical Company Ltd.

Author information

Authors and Affiliations

  1. Department of Hematopoietic Factors, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai Minato-ku, Tokyo, 108-8639, Japan
    S Morita, T Kojima & T Kitamura

Authors

  1. S Morita
    You can also search for this author inPubMed Google Scholar
  2. T Kojima
    You can also search for this author inPubMed Google Scholar
  3. T Kitamura
    You can also search for this author inPubMed Google Scholar

Rights and permissions

About this article

Cite this article

Morita, S., Kojima, T. & Kitamura, T. Plat-E: an efficient and stable system for transient packaging of retroviruses.Gene Ther 7, 1063–1066 (2000). https://doi.org/10.1038/sj.gt.3301206

Download citation

Keywords

This article is cited by