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.

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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.

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  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

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  1. S Morita
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  2. T Kojima
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  3. T Kitamura
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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

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