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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 Iba^1,*

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 (Neo^r) 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 Neo^r expression. By inserting an mRNA-destabilizing signal into the 3' untranslated region of the Neo^r gene to reduce the amount of Neo^r 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 -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.

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^* 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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