A novel family of retroviral vectors for the rapid production of complex stable cell lines
A novel family of retroviral vectors for the rapid production of complex stable cell lines
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DOI：
10.1006/abio.2001.5327
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发表时间：
2001-10-01
影响因子：
2.9
通讯作者：
Marrack, P
中科院分区：
文献类型：
--
作者：
Schaefer, BC;Mitchell, TC;Marrack, P
稳定细胞系的产生是细胞生物学中的一项重要技术，并且通常是涉及新基因或基因突变功能表征的研究中的限速步骤。为了促进这一过程，产生了一个新的逆转录病毒载体家族，即 pE 载体家族。 pE 载体中的逆转录病毒序列取自莫洛尼鼠白血病病毒 (MMLV) 载体 pMFG，该载体已被证明比前几代 MMLV 载体更一致、更高水平地表达 cDNA 插入片段。这些载体含有四种不同的内部核糖体进入位点选择标记，可以高效选择表达所需 cDNA 的转导子。 pE 载体采用附加型设计，可以长期生产高滴度病毒，而无需亚克隆生产线。使用组合感染和组合药物选择的策略，我们证明 pE 载体可用于在不到 2 周的时间内生成表达至少三种新型 cDNA 的稳定的多克隆细胞系。因此，这些载体的使用将极大地加速复杂稳定细胞系的产生。 (C) 2001 年学术出版社。
The production of stable cell lines is an important technique in cell biology, and it is often the rate-limiting step in studies involving the characterization of the function of novel genes or gene mutations. To facilitate this process, a novel family of retroviral vectors, the pE vector family, has been generated. The retroviral sequences in the pE vectors have been taken from the Moloney murine leukemia virus (MMLV) vector pMFG, which has been shown to express cDNA inserts more consistently and at higher levels than earlier generations of MMLV vectors. These vectors contain four different internal ribosome entry site-selectable markers, allowing high-efficiency selection of transductants expressing the desired cDNA. The pE vectors have an episomal design to allow long-term production of high-titer virus without the need for subcloning the producer line. Using a strategy of combinatorial infection followed by combinatorial drug selection, we demonstrate that the pE vectors can be used to generate stable, polyclonal cell lines expressing at least three novel cDNAs in less than 2 weeks. The use of these vectors will thus dramatically accelerate the production of complex stable cell lines. (C) 2001 Academic Press.