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首页> 外文期刊>Molecular and Cellular Biology >Construction of a defective retrovirus containing the human hypoxanthine phosphoribosyltransferase cDNA and its expression in cultured cells and mouse bone marrow.
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Construction of a defective retrovirus containing the human hypoxanthine phosphoribosyltransferase cDNA and its expression in cultured cells and mouse bone marrow.

机译：含有人次黄嘌呤磷酸核糖基转移酶cDNA的缺陷型逆转录病毒的构建及其在培养细胞和小鼠骨髓中的表达。

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Defective ecotropic and amphotropic retroviral vectors containing the cDNA for human hypoxanthine phosphoribosyltransferase (HPRT) were developed for efficient gene transfer and high-level cellular expression of HPRT. Helper cell clones which produced a high viral titer were generated by a simplified method which minimizes cell culture. We used the pZIP-NeoSV(X) vector containing a human hprt cDNA. Viral titers (1 X 10(3) to 5 X 10(4)/ml) of defective SVX HPRT B, a vector containing both the hprt and neo genes, were increased 3- to 10-fold by cocultivation of the ecotropic psi 2 and amphotropic PA-12 helper cells. Higher viral titers (8 X 10(5) to 7.5 X 10(6] were obtained when nonproducer NIH 3T3 cells or psi 2 cells carrying a single copy of SVX HPRT B were either transfected or infected by Moloney leukemia virus. The SVX HPRT B defective virus partially corrected the HPRT deficiency (4 to 56% of normal) of cultured rodent and human Lesch-Nyhan cells. However, instability of HPRT expression was detected in several infected clones. In these unstable variants, both retention and loss of the SVX HPRT B sequences were observed. In the former category, cells which became HPRT- (6-thioguanine resistant [6TGr]) also became G418s, indicative of a cis-acting down regulation of expression. Both hypoxanthine-aminopterin-thymidine resistance (HATr) and G418r could be regained by counterselection in hypoxanthine-aminopterin-thymidine. In vitro mouse bone marrow experiments indicated low-level expression of the neo gene in in vitro CFU assays. Individual CFU were isolated and pooled, and the human hprt gene was shown to be expressed. These studies demonstrated the applicability of vectors like SVX HPRT B for high-titer production of defective retroviruses required for hematopoietic gene transfer and expression.
机译：开发了含有人类次黄嘌呤磷酸核糖基转移酶（HPRT）cDNA的缺陷型嗜性和两性逆转录病毒载体，用于高效基因转移和HPRT的高水平细胞表达。产生高病毒滴度的辅助细胞克隆是通过使细胞培养最小化的简化方法产生的。我们使用了包含人hprt cDNA的pZIP-NeoSV（X）载体。有缺陷的SVX HPRT B（一种同时包含hprt和neo基因的载体）的病毒滴度（1 X 10（3）至5 X 10（4）/ ml）通过共生psi 2共同培养可提高3至10倍和两性的PA-12辅助细胞。当携带单拷贝SVX HPRT B的非生产性NIH 3T3细胞或psi 2细胞被莫洛尼白血病病毒转染或感染时，可获得更高的病毒滴度（8 X 10（5）至7.5 X 10（6]）。缺陷病毒可以部分纠正培养的啮齿动物和人类Lesch-Nyhan细胞的HPRT缺陷（正常水平的4％至56％），但是，在几个受感染的克隆中检测到HPRT表达不稳定，在这些不稳定的变体中，SVX的保留和丢失观察到HPRT B序列，在前一类中，变为HPRT-（6-硫代鸟嘌呤抗性[6TGr]）的细胞也变为G418s，表明其顺式作用下调了表达。两个黄嘌呤-氨基蝶呤-胸苷抗性（HATr）通过在次黄嘌呤-氨基蝶呤-胸腺嘧啶核苷中进行反选择可以重新获得G418r和G418r;体外小鼠骨髓实验表明，在体外CFU测定中neo基因的表达水平很低;分离并合并了单个CFU，以及人hprt基因被证明是表达出来的。这些研究证明了诸如SVX HPRT B之类的载体可用于高滴度生产造血基因转移和表达所需的缺陷性逆转录病毒。

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