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A versatile nonviral vector system for tetracycline-dependent one-step conditional induction of transgene expression

Gene Therapy volume 16, pages 1383–1394 (2009)Cite this article

Abstract

In this study, we describe a novel self-contained, nonviral vector system for the rapid development of tetracycline (Tet)-inducible transgene expression systems in mammalian cell lines. To avoid multiple rounds of clonal selection for the establishment of stably transfected cell clones, as is necessary with conventional systems, we constructed a multicomplementary DNA(cDNA) expression vector that enables both one-step targeted genomic integration and conditional induction of transgene expression. This vector system consists of several modules including a Tet-inducible promoter directing the expression of a transgene and two Tet repressor expression units placed in tandem on a single vector. The cell clones, generated using a one-step φC31 integrase-mediated chromosomal integration of the multi-cDNA expression construct, showed a stable and robust expression with high induction rates upon addition of doxycycline inducer in five different cell lines tested. By using this system, we show c-Src-induced cell transformation and anticancer cell therapy for this transformation in cultured fibroblast cells. The results show a rapid production and accumulation of target protein on addition of the inducer starting from extremely low background levels and reduction to background levels in a matter of days after the inducer was withdrawn from the culture medium.

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Acknowledgements

We are grateful to Dr Michele P Calos for pCMV-phiC31 Int and pTA-attB, Dr Atsushi Miyawaki, Riken (Japan) for providing pd1-Venus and Dr Gary Felsenfeld for pJC5-4. This work was supported in part by the Grant-in-Aid for Scientific Research from The New Energy and Industrial Technology Development Organization, Japan (NEDO). Gateway, Max Efficiency and Library Efficiency are registered trademarks of Invitrogen Corp. Clonase, pDONR, DH10B, DB3.1, pENTR and pDEST, ChargeSwitch Total RNA cell kit, and Superscript First-strand Synthesis System are trademarks of Invitrogen Corp.

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Authors and Affiliations

  1. Department of Molecular Biology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan,

    K Inoue, T Sone, F Nishiumi, H Kishine, Y Sasaki, T Andoh & F Imamoto

  2. Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan,

    C Oneyama & M Okada

  3. Invitrogen Corporation, R&D, Carlsbad, CA, USA

    J D Chesnut

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  1. K Inoue
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  2. T Sone
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  3. C Oneyama
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Correspondence to F Imamoto.

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Supplementary Information accompanies the paper on Gene Therapy website (http://www.nature.com/gt)

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Inoue, K., Sone, T., Oneyama, C. et al. A versatile nonviral vector system for tetracycline-dependent one-step conditional induction of transgene expression. Gene Ther 16, 1383–1394 (2009). https://doi.org/10.1038/gt.2009.105

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