The pCL vector system: Rapid production of helper-free, high-titer, recombinant retroviruses
The pCL vector system: Rapid production of helper-free, high-titer, recombinant retroviruses
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DOI：
10.1128/jvi.70.8.5701-5705.1996
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发表时间：
1996-08-01
影响因子：
5.4
通讯作者：
Verma, IM
中科院分区：
文献类型：
--
作者：
Naviaux, RK;Costanzi, E;Verma, IM
我们描述了逆转录病毒载体和包装质粒的构建和表征，这些载体和包装质粒在48小时内产生滴度为1 × 10（6）至5 × 10（6）的无辅助逆转录病毒。这些载体含有人巨细胞病毒增强子-启动子的立即早期区，其在5 ′ U3区的TATA盒处与莫洛尼鼠白血病病毒长末端重复序列融合，产生pCL启动子。通过选择设计用于表达来自四种启动子（二氢叶酸还原酶、劳斯肉瘤病毒、长末端重复序列或巨细胞病毒）之一的基因的载体，pCL系统允许研究者将靶细胞中的基因表达水平控制在100倍范围内，同时保持来自瞬时转染的生产细胞的病毒的均匀高滴度。pCL包装质粒缺乏包装信号（Delta Psi），并且包括增加的安全性修饰，该修饰通过缺失3' U3增强子使它们自失活。嗜亲性，嗜亲性（4070 A），和嗜亲性水貂细胞灶形成杂交（10A 1）信封建设已准备和测试，允许灵活选择载体假型根据实验需要。载体上清液不含辅助病毒，并且在293细胞中瞬时转染的2天内具有足够高的滴度，以允许感染培养物中超过50%的随机循环靶细胞。我们通过使用这些载体将三种有效的细胞周期控制基因（p16（INK 4A），p53和Rbl基因）转移到人胶质母细胞瘤细胞中来证明这些载体的功效。
We describe the construction and characterization of retroviral vectors and packaging plasmids that produce helper-free retrovirus with titers of 1 x 10(6) to 5 x 10(6) within 48 h. These vectors contain the immediate early region of the human cytomegalovirus enhancer-promoter fused to the Moloney murine leukemia virus long terminal repeat at the TATA box in the 5' U3 region, yielding the pCL promoter. By selecting vectors designed to express genes from one of four promoters (dihydrofolate reductase, Rous sarcoma virus, long terminal repeat, or cytomegalovirus), the pCL system permits the investigator to control the level of gene expression in target cells over a 100-fold range, while maintaining uniformly high titers of virus from transiently transfected producer cells. The pCL packaging plasmids lack a packaging signal (Delta Psi) and include an added safety modification that renders them self-inactivating through the deletion of the 3' U3 enhancer. Ecotropic, amphotropic (4070A), and amphotropic-mink cell focus-forming hybrid (10A1) envelope constructions have been prepared and tested, permitting flexible selection of vector pseudotype in accordance with experimental needs. Vector supernatants are free of helper virus and are of sufficiently high titer within 2 days of transient transfection in 293 cells to permit infection of more than 50% of randomly cycling target cells in culture. We demonstrated the efficacy of these vectors by using them to transfer three potent cell cycle control genes (the p16(INK4A), p53, and Rbl genes) into human glioblastoma cells.