Bicistronic and two-gene retroviral vectors for using MDR1 as a selectable marker and a therapeutic gene.
Bicistronic and two-gene retroviral vectors for using MDR1 as a selectable marker and a therapeutic gene.
复制标题
使用 MDR1 作为选择标记和治疗基因的双顺反子和双基因逆转录病毒载体。
作者：
Metz,MZ;Matsumoto,L;Winters,KA;Doroshow,JH;Kane,SE
我们描述了一系列双基因和双顺反子逆转录病毒载体，其使用humanMDR 1基因作为在转导细胞中过表达第二异源基因的选择标记。所述载体使用哈维鼠肉瘤病毒序列用于病毒表达和包装功能，并且包括在内部启动子（双基因载体）或内部核糖体进入位点（双顺反子载体）的控制下克隆感兴趣的外源基因的位点。为了表征这些载体，我们使用neo作为外源基因表达的报告基因，并作为与MDR 1比较的独立选择标记。每种载体构建支持小鼠和人细胞系的高滴度逆转录病毒产生和转导。使用MDR 1-neovirus上清液进行平行滴度测定，我们发现基于秋水仙素抗性的滴度比基于G418抗性的滴度低10- 20倍，这表明在NIH 3 T3和KB-3-1细胞系中MDR 1是比neovirus更严格的选择标记。尽管双基因载体的新基因表达受到宿主特异性内部启动子活性的限制，但双顺反子载体在测试的三种细胞系中具有高度活性。在K562细胞中，使用双顺反子载体，用秋水仙素选择导致至少20倍高的表达theMDR 1基因产物比G418选择，表明stringentMDR 1选择系统是非常有效的获得外源基因的过表达。逆转录病毒载体携带MDR 1作为一个选择标记加上第二个异源目的基因，可以广泛应用于体外和体内基因转移技术的应用，包括基因治疗。
We describe a series of two-gene and bicistronic retroviral vectors that use the humanMDR1 gene as a selectable marker for the overexpression of a second heterologous gene in transduced cells. The vectors use Harvey murine sarcoma virus sequences for viral expression and packaging functions and include sites for cloning foreign genes of interest under the control of either an internal promoter (two-gene vectors) or an internal ribosome entry site (bicistronic vectors). To characterize these vectors, we usedneoas a reporter gene for foreign gene expression and as an independently selectable marker for comparison withMDR1. Each of the vector constructions supported high-titer retrovirus production and transduction of mouse and human cell lines. UsingMDR1–neovirus supernatants in parallel titering assays, we found that titers based on colchicine resistance were 10- to 20-fold lower than titers based on G418 resistance, suggesting thatMDR1 is a more stringent selectable marker thanneoin NIH 3T3 and KB-3-1 cell lines. Whereasneogene expression with the two-gene vectors was subject to host-specific limitations on internal promoter activity, the bicistronic vectors were highly active in three cell lines tested. In K562 cells, using the bicistronic vector, selection with colchicine led to at least 20-fold higher expression of theMDR1 gene product than did selection with G418, suggesting that the stringentMDR1 selection system is very efficient for obtaining overexpression of foreign genes. Retroviral vectors carryingMDR1 as a selectable marker plus a second, heterologous gene of interest could have widespread utility forin vitroandin vivoapplications of gene transfer technology, including gene therapy.