A new retrovirus packaging cell for gene transfer constructed from amplified long terminal repeat-free chimeric proviral genes.
The retroviral gene transfer system is a powerful tool for somatic gene therapy. A retroviral stock with a high viral titer and lacking replication-competent virus (RCV) is desirable for this type of gene transfer. To fulfill these requirements, we made a new packaging cell line, designated ampli-GPE. To reduce the homology between proviral DNA in the packaging cell and retroviral vector, the gag-pol and env genes of Moloney murine leukemia virus were separated onto two different plasmids, pGP-KV and pENV-KV, respectively, in which the 5' long terminal repeat and the 3' long terminal repeat had been replaced by the mouse metallothionein I promoter or the human beta-globin gene containing the polyadenylation site as control units for the gag-pol and env genes. In addition, these plasmids contained 69% of the bovine papillomavirus gene for gene amplification to obtain production of virus at a high titer. NIH 3T3 clones containing approximately 20 to 50 copies of the gag-pol and env genes were selected and designated ampli-GPE. When ampli-GPE was transfected with the N2 vector or pZipNeoSV(DHFR) derived from pZipNeoSV(X)1, we established clones producing titers of 5 x 10(6) and 1 x 10(6) CFU/ml, respectively. There was no sign of RCV generation in any virus-producing cells from ampli-GPE. However, virus-producing cells derived from psi 2 cells transfected with N2 did generate RCV. Thus, we showed that ampli-GPE, possessing the minimum complement of proviral genes, has potential for the development of a gene transfer system.
Takahara
Y
Y
Central Research Laboratories, Ajinomoto Co., Ltd., Kawasaki, Japan.
Hamada
K
K
Housman
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DE
D016475:3T3 Cells; D000818:Animals; D004274:DNA, Recombinant / Q000235:genetics; D015685:Fusion Proteins, gag-pol / Q000235:genetics; D005784:Gene Amplification; D015752:Genes, env / Q000235:genetics; D015750:Genes, gag / Q000235:genetics; D015751:Genes, pol / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics*; D005914:Globins / Q000235:genetics; D008668:Metallothionein / Q000235:genetics; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics*; D027383:Papillomaviridae / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011533:Proviruses / Q000235:genetics*; D011817:Rabbits; D014162:Transfection / Q000235:genetics*; D014779:Virus Replication
D004274:DNA, Recombinant; D015685:Fusion Proteins, gag-pol; D005914:Globins; D008668:Metallothionein