Inducible, high-level production of infectious murine leukemia retroviral vector particles pseudotyped with vesicular stomatitis virus G envelope protein.
Inducible, high-level production of infectious murine leukemia retroviral vector particles pseudotyped with vesicular stomatitis virus G envelope protein.
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DOI：
10.1089/hum.1995.6.9-1203
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发表时间：
1995-09
影响因子：
4.2
通讯作者：
Jean Yee Hwa Yang;E. Vanin;M. Whitt;M. Fornerod;R. Zwart;R. Schneiderman;G. Grosveld;A. Nienhuis
中科院分区：
文献类型：
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作者：
Jean Yee Hwa Yang;E. Vanin;M. Whitt;M. Fornerod;R. Zwart;R. Schneiderman;G. Grosveld;A. Nienhuis
鼠白血病病毒（MuLV）已被改造用作实验和人类基因治疗应用的基因转移载体。它们用于这些目的的效用受到有限的宿主范围和可用生产克隆的相对低滴度的限制。在产生MuLV Gag和Pol蛋白的细胞中瞬时表达的具有水泡性口炎病毒包膜蛋白（VSV-G）的MuLV颗粒的假型化已经产生了具有更宽宿主范围的载体制剂，其可以通过超离心浓缩。我们已经探索了使用类固醇诱导型和四环素调节的启动子系统（因为VSV-G蛋白在组成型表达时对细胞有毒，所以是必要的）来获得能够大量生产VSV-G假型MuLV颗粒的稳定生产细胞系。一种能表达嵌合转录因子的包装细胞系和生产克隆，所述嵌合转录因子由四环素阻遏物（tetR）和单纯疱疹病毒VP 16基因的VP 16反式激活序列组成，并含有与最小启动子连接的VSV-G编码序列，所述最小启动子具有四环素应答操纵子（tetO）的七个串联拷贝，当在不存在四环素的情况下培养时，显示出高水平的VSV-G蛋白表达。以10（5）-10（6）感染性菌落形成单位/ml（cfu/ml）的滴度产生的载体颗粒可以通过超离心有效地浓缩，产生具有10（9）cfu/ml滴度的载体制剂。这些细胞株生长正常时，VSV-G蛋白表达抑制四环素。这样的生产者克隆为未来的人类基因治疗应用带来了希望。
Murine leukemia viruses (MuLV) have been adapted for use as gene transfer vectors for experimental and human gene therapy applications. Their utility for these purposes has been circumscribed by the limited host range and relatively low titer of available producer clones. Pseudotyping of MuLV particles with the vesicular stomatitis virus envelope protein (VSV-G), expressed transiently in cells producing MuLV Gag and Pol proteins, has yielded vector preparations with a broader host range that can be concentrated by ultracentrifugation. We have explored the use of steroid-inducible and tetracycline-modulated promoter systems (necessary because the VSV-G protein is toxic to cells when constitutively expressed) to derive stable producer cell lines capable of substantial production of VSV-G pseudotyped MuLV particles. A packaging cell line and producer clones capable of expressing a chimeric transcription factor, composed of the tetracycline repressor (tetR) and the VP16 trans-activating sequences of herpes simplex virus VP16 gene and containing the VSV-G coding sequences linked to a minimal promoter having seven tandem copies of the tetracycline responsive operator (tetO), exhibited high levels of VSV-G protein expression when cultured in the absence of tetracycline. Vector particles, produced at titers of 10(5)-10(6) infectious colony forming units per ml (cfu/ml), could be concentrated effectively by ultracentrifugation yielding vector preparations having a titer of 10(9) cfu/ml. These cell lines grew normally when VSV-G protein expression was repressed by tetracycline. Such producer clones hold promise for future human gene therapy applications.