title: 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.
abstract: 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 beta-galactosidase. They reproducibly produced high-titer virus stocks of VSV-G-pseudotyped retrovirus (1.0 x 10(6) 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.
authors.affiliation: Department of Gene Regulation, Institute of Medical Science, University of Tokyo, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Arai
authors.forename: K
authors.initials: K
authors.lastname: Matsumoto
authors.forename: K
authors.initials: K
authors.lastname: Saitoh
authors.forename: M
authors.initials: M
authors.lastname: Ui
authors.forename: T
authors.initials: T
authors.lastname: Ito
authors.forename: M
authors.initials: M
authors.lastname: Murakami
authors.forename: Y
authors.initials: Y
authors.lastname: Kanegae
authors.forename: I
authors.initials: I
authors.lastname: Saito
authors.forename: F L
authors.initials: FL
authors.lastname: Cosset
authors.forename: Y
authors.initials: Y
authors.lastname: Takeuchi
authors.forename: H
authors.initials: H
authors.lastname: Iba
mesh_terms: D000818:Animals; D002460:Cell Line; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D019426:Integrases / Q000235:genetics*; D008562:Membrane Glycoproteins; D051379:Mice; D012190:Retroviridae / Q000235:genetics*; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics*; D014759:Viral Envelope Proteins / Q000235:genetics*; D014764:Viral Proteins
chemical_list: C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases