Aran J M, Gottesman M M, Pastan I
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3176-80. doi: 10.1073/pnas.91.8.3176.
Bicistronic cassettes under control of a single promoter have recently been suggested as useful tools for coordinate expression of two different foreign proteins in mammalian cells. Using the long 5' untranslated region of encephalomyocarditis virus as translational enhancer of the second gene, a bicistronic unit composed of cDNA for human P-glycoprotein [the product of the multidrug resistance gene, MDR1 (also called PGY1)] as selectable marker and cDNA for human glucocerebrosidase (GC; EC 3.2.1.45) (a membrane-associated lysosomal hydrolase) was constructed. NIH 3T3 cells transfected with a Harvey murine sarcoma virus retroviral vector carrying this bicistronic cassette (pHaMCG) express active P-glycoprotein and GC and expression of both proteins augments coordinately with selection for increased colchicine resistance. Percoll gradient analysis of homogenates showed that GC was targeted to the lysosomal fraction. The ability to select for expression of GC with natural product drugs after introduction of the pHaMCG retroviral vector may be useful in gene therapy strategies for Gaucher disease.
最近，由单个启动子控制的双顺反子盒被认为是在哺乳动物细胞中协调表达两种不同外源蛋白的有用工具。利用脑心肌炎病毒的长5'非翻译区作为第二个基因的翻译增强子，构建了一个双顺反子单元，其由作为选择标记的人P-糖蛋白[多药耐药基因MDR1（也称为PGY1）的产物]的cDNA和人葡糖脑苷脂酶（GC；EC 3.2.1.45）（一种膜相关溶酶体水解酶）的cDNA组成。用携带此双顺反子盒（pHaMCG）的哈维鼠肉瘤病毒逆转录病毒载体转染的NIH 3T3细胞表达活性P-糖蛋白和GC，并且两种蛋白的表达随着对秋水仙碱抗性增加的选择而协同增强。匀浆的Percoll梯度分析表明，GC靶向溶酶体部分。在引入pHaMCG逆转录病毒载体后，利用天然产物药物选择GC表达的能力可能在戈谢病的基因治疗策略中有用。
