A NEW RETROVIRUS PACKAGING CELL FOR GENE-TRANSFER CONSTRUCTED FROM AMPLIFIED LONG TERMINAL REPEAT-FREE CHIMERIC PROVIRAL GENES

被引:21
作者
TAKAHARA, Y [1 ]
HAMADA, K [1 ]
HOUSMAN, DE [1 ]
机构
[1] MIT,DEPT BIOL,CTR CANC RES,CAMBRIDGE,MA 02139
关键词
D O I
10.1128/JVI.66.6.3725-3732.1992
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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plasmids, pGP-KV and pENV-KV, respectively, in which the 5' long terminal repeat and the 3' long terminal repeat had been replaced by the mouse metallothionein I promoter or the human beta-globin gene containing the polyadenylation site as control units for the gag-pol and env genes.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.
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页码:3725 / 3732
页数:8
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