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J Virol, February 1998, p. 1115-1121, Vol. 72, No. 2
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

A New System for Stringent, High-Titer Vesicular Stomatitis Virus G Protein-Pseudotyped Retrovirus Vector Induction by Introduction of Cre Recombinase into Stable Prepackaging Cell Lines

Tohru Arai,1,2 Kazuyuki Matsumoto,1 Kanako Saitoh,1 Motoyasu Ui,1 Taiji Ito,1 Masao Murakami,1 Yumi Kanegae,3 Izumu Saito,3 François-Loïc Cosset,4 Yasuhiro Takeuchi,5 and Hideo Iba1,*

Department of Gene Regulation1 and Laboratory of Molecular Genetics,3 Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108, and Tsukuba Research Laboratories, Eisai Co., Ltd., Tsukuba-shi, Ibaraki 300-26,2 Japan; CGMC, CNRS UMR106, Universite Claude Bernard Lyon-1, 69622 Villeurbanne Cedex, France4; and Institute of Cancer Research, Chester Beatty Laboratories, London SW3 6JB, United Kingdom5

Received 16 June 1997/Accepted 21 October 1997

We report here on stable prepackaging cell lines which can be converted into packaging cell lines for high-titer vesicular stomatitis virus G protein (VSV-G)-pseudotyped retrovirus vectors by the introduction of Cre recombinase-expressing adenovirus. The generated prepackaging cell lines constitutively express the gag-pol genes and contain an inducible transcriptional unit for the VSV-G gene. From this unit, the introduced Cre recombinase excised both a neomycin resistance (Neor) gene and a poly(A) signal flanked by a tandem pair of loxP sequences and induced transcription of the VSV-G gene from the same promoter as had been used for Neor expression. By inserting an mRNA-destabilizing signal into the 3' untranslated region of the Neor gene to reduce the amount of Neor transcript, we were able efficiently to select the clones capable of inducing VSV-G at high levels. Without the introduction of Cre recombinase, these cell lines produce neither VSV-G nor any detectable infectious virus at all, even after the transduction of a murine leukemia virus-based retrovirus vector encoding beta -galactosidase. They reproducibly produced high-titer virus stocks of VSV-G-pseudotyped retrovirus (1.0 × 106 infectious units/ml) from 3 days after the introduction of Cre recombinase. We also present evidence that VSV-G-producing cells are still fully susceptible to transduction by VSV-G pseudotypes. However, in this vector-producing system, which regulates VSV-G pseudotype production in an all-or-none manner, the integration of vector DNA into packaging cell lines would be minimized. We further show that heparin significantly inhibits retransduction of VSV-G pseudotypes in the culture fluids of packaging cell lines, leading to a two- to fourfold increase in the yield of the pseudotypes after induction. This vector-producing system was very stable and should be advantageous in human gene therapy.

* Corresponding author. Mailing address: Department of Gene Regulation, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan. Phone: 81-3-5449-5730. Fax: 81-3-5449-5449. E-mail: iba{at}hgc.ims.u-tokyo.ac.jp.




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