CONSTRUCTION AND USE OF A SAFE AND EFFICIENT AMPHOTROPIC PACKAGING CELL-LINE
CONSTRUCTION AND USE OF A SAFE AND EFFICIENT AMPHOTROPIC PACKAGING CELL-LINE
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DOI：
10.1016/0042-6822(88)90101-8
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发表时间：
1988-12-01
期刊：
影响因子：
3.7
通讯作者：
BANK, A
中科院分区：
文献类型：
--
作者：
MARKOWITZ, D;GOFF, S;BANK, A
构建了两性性逆转录病毒包装细胞系，将辅助病毒的gag、pol和env基因分离在两个不同的质粒上，去除包装信号和3”长末端重复序列。为此，将含有Moloney小鼠白血病病毒gag和pol基因的质粒转染到NIH 3T3细胞中，并将含有4070A两性环境基因的质粒转染到产生高水平逆转录酶的克隆之一中。然后分离出一个产生高水平两性性env蛋白（GP+envAm12）的克隆。当转染到GP+envAm12细胞时，含有新霉素耐药基因的逆转录病毒载体N2在3T3细胞上的滴度从1.3倍到102至106 CFU/ml不等。104到2.7倍。HeLa细胞105 CFU/ml，从1.0。102到6.0倍。103 CFU/ml对K562细胞进行G418抗性检测。这些滴度与使用PA317细胞系获得的滴度相当。GP+envAm12包装线的安全性测试显示，没有证据表明会产生野生型病毒。因此，GP+enAm12细胞系在基因转移到人细胞中的效率和安全性可能为人类基因治疗的实验提供了一个最佳的系统。
An amphotropic retrovirus packaging cell line was constructed in which the gag, pol, and env genes of the helper virus are separated on two different plasmids and in which the packaging signals and 3'' long terminal repeats are removed. To do this, a plasmid containing the Moloney murine leukemia virus gag and pol genes was transfected into NIH 3T3 cells, and a plasmid containing the 4070A amphotropic env gene was transfected into one of the resulting clones which produced a high level of reverse transcriptase. A clone producing a high level of amphotropic env protein (GP+envAm12) was then isolated. When transfected into GP+envAm12 cells, titers of the retroviral vector N2, containing a neomycin resistance gene, ranged from 102 to > 106 CFU/ml on 3T3 cells, from 1.3 .times. 104 to 2.7 .times. 105 CFU/ml on HeLa cells, and from 1.0 .times. 102 to 6.0 .times. 103 CFU/ml on K562 cells when assayed by G418 resistance. These titers were comparable to titers obtained using the PA317 cell line. Tests for the safety of the GP+envAm12 packaging line showed no evidence for the generation of wild-type virus. Thus, the efficiency and safety of the GP+enAm12 cell line in gene transfer into human cells may provide an optimal system for experiments whose goal is human gene therapy.