Overcoming interference to retroviral superinfection results in amplified expression and transmission of cloned genes.
Overcoming interference to retroviral superinfection results in amplified expression and transmission of cloned genes.
复制标题
克服逆转录病毒重复感染的干扰会导致克隆基因的表达和传播放大。
DOI：
10.1073/pnas.85.15.5404
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发表时间：
1988
影响因子：
11.1
通讯作者：
Kabat,D
中科院分区：
文献类型：
--
作者：
Bestwick,RK;Kozak,SL;Kabat,D
描述了在脊椎动物细胞中高水平稳定表达克隆基因并在高滴度病毒制剂中获得这些基因的程序。该过程使用逆转录病毒载体和两种“包装细胞系”的混合物，将逆转录病毒基因组整合到具有不同宿主范围包膜的病毒体中。在这些共培养物中，重复感染的干扰屏障被克服，逆转录病毒载体可以在没有传染性辅助病毒的情况下复制，并且细胞被含有克隆基因的原病毒的多个拷贝感染。该程序用于扩增 Friend 脾病灶形成病毒（一种在其他细胞培养物中存在复制缺陷的逆转录病毒）编码的膜糖蛋白的表达。在 DNA 原病毒、RNA 和蛋白质水平上测量扩增。此外，将人类生长激素基因插入逆转录病毒载体中，我们观察到生长激素合成和分泌的扩增。免疫印迹分析表明，扩增的生长激素经过适当处理。描述了一种在共培养扩增中异常活跃的载体（pSFF）。
A procedure is described for stably expressing cloned genes at high levels in vertebrate cells and for obtaining these genes in high-titer virus preparations. The process uses retroviral vectors and mixtures of two "packaging cell lines" that incorporate retroviral genomes into virions with different host-range envelopes. In these cocultures, interference barriers to superinfection are overcome, retroviral vectors can replicate in the absence of a transmissible helper virus, and the cells become infected with multiple copies of the provirus that contains the cloned gene. This procedure was used to amplify expression of the membrane glycoprotein that is encoded by Friend spleen focus-forming virus, a retrovirus that is replication defective in other cell cultures. Amplifications were measured at the DNA provirus, RNA, and protein levels. In addition, the human growth hormone gene was inserted into retroviral vectors and we observed amplifications of growth hormone synthesis and secretion. The amplified growth hormone was properly processed as indicated by immunoblot analyses. A vector is described (pSFF) that is exceptionally active in coculture amplification.