A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes
A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes
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DOI：
10.1073/pnas.93.21.11400
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发表时间：
1996-10-15
影响因子：
11.1
通讯作者：
Mulligan, RC
中科院分区：
文献类型：
--
作者：
Ory, DS;Neugeboren, BA;Mulligan, RC
我们已经产生了一种人类293衍生的逆转录病毒包装细胞系（293GPG），能够产生高滴度的重组Moloney小鼠白血病病毒颗粒，其中包含水泡性口炎病毒G （VSV-G）蛋白。为了实现逆转录病毒gag-pol多蛋白的表达，将gag-pol的精确编码序列引入到完全利用非逆转录病毒信号进行基因表达的载体中。由于VSV-G蛋白的组成表达在293细胞中是有毒的，我们使用tet(R)/VP 16反激活子和tet(o)最小启动子系统来诱导四环素可调节的VSV-G表达。用MFG稳定转染293GPG包装细胞系后。利用SnlsLacZ逆转录病毒载体构建，可以很容易地分离出滴度接近10(7)个集落形成单位/ml的稳定产病毒细胞系。使用修饰版MFG瞬时转染293GPG细胞。SnlsLacZ使用巨细胞病毒IE启动子驱动原病毒基因组的转录，导致滴度接近10(6)个集落形成单位/ml。使用293GPG细胞生成的逆转录病毒/VSV-G假型对人类补体的抗性明显高于常用的两性性载体，并且可以高度浓缩（约1000倍）。这种新的包装细胞系可能被证明对评估逆转录病毒载体在体内直接基因转移的潜在用途特别有用。在可能释放具有复制能力的病毒方面，这种细胞系的设计至少在理论上比现有的细胞系有优势。
We have generated a human 293-derived retroviral packaging cell line (293GPG) capable of producing high titers of recombinant Moloney murine leukemia virus particles that have incorporated the vesicular stomatitis virus G (VSV-G) protein. To achieve expression of the retroviral gag-pol polyprotein, the precise coding sequences for gag-pol were introduced into a vector which utilizes totally nonretroviral signals for gene expression. Because constitutive expression of the VSV-G protein is toxic in 293 cells, we used the tet(R)/VP 16 transactivator and tet(o) minimal promoter system for inducible, tetracycline-regulatable expression of VSV-G. After stable transfection of the 293GPG packaging cell line with the MFG.SnlsLacZ retroviral vector construct, it was possible to readily isolate stable virus-producing cell lines with titers approaching 10(7) colony-forming units/ml. Transient transfection of 293GPG cells using a modified version of MFG.SnlsLacZ, in which the cytomegalovirus IE promoter was used to drive transcription of the proviral genome, led to titers of approximate to 10(6) colony-forming units/ml. The retroviral/VSV-G pseudotypes generated using 293GPG cells were significantly more resistant to human complement than commonly used amphotropic vectors and could be highly concentrated (> 1000-fold). This new packaging cell line may prove to be particularly useful for assessing the potential use of retroviral vectors for direct in vivo gene transfer. The design of the cell line also provides at least theoretical advantages over existing cell lines with regard to the possible release of replication-competent virus.