利用两个内部核糖体进入位点构建和表征单转录三反转录病毒载体。

M Z Metz, A Pichler, K Kuchler, S E Kane
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引用次数: 17

摘要

我们描述了一系列包含三个基因共转录的两个内部核糖体进入位点(IRES)的逆转录病毒载体。Harvey小鼠肉瘤病毒长末端重复序列控制单转录三反子mRNA的转录。5'端最开放的阅读框受到帽区依赖或帽区独立的翻译控制,而下游的两个开放阅读框则使用各自IRES元件的起始密码子以帽区独立的方式翻译。两种IRES元素均取自脑心肌炎病毒。为了对这些载体进行表征,我们将人类多药耐药基因(MDR1)置于5′位置,绿色荧光蛋白(GFP)基因置于中间位置,neo置于3′位置。将载体直接转染到NIH3T3小鼠成纤维细胞或包装成逆转录病毒后转导到NIH3T3细胞。基因转移后,用秋水仙碱选择表达MDR1基因,或用G418选择表达neo基因。因此,我们可以在选择5'-most或3'-most基因的条件下确定三分子载体的功能。在dna介导的转染中,我们能够在任何选择条件下实现所有三个开放阅读框的表达。使用秋水仙碱选择MDR1表达时,我们获得了比使用G418选择neo表达时更高的三个基因表达。未选择的GFP基因(中间顺子)的表达不稳定,很可能是由于在长期培养过程中丢失了完整的GFP DNA序列。我们能够实现逆转录病毒介导的所有三个基因的转导,但这是一个低效的过程。
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Construction and characterization of single-transcript tricistronic retroviral vectors using two internal ribosome entry sites.

We describe a series of retroviral vectors containing two internal ribosome entry sites (IRES) for the co-transcription of three genes. Transcription of the single-transcript tricistronic mRNA is under the control of a Harvey murine sarcoma virus long terminal repeat. The 5'-most open reading frame is under either cap-dependent or cap-independent translational control, while the two downstream open reading frames are translated in a cap-independent fashion using the initiation codons of their respective IRES elements. Both IRES elements are taken from the encephalomyocarditis virus. To characterize these vectors, we used the human multidrug resistance gene (MDR1) in the 5' position, the gene for green fluorescent protein (GFP) in the middle position, and neo in the 3' position. The vectors were either transfected directly into NIH3T3 mouse fibroblasts or packaged into retrovirus and then transduced into NIH3T3 cells. Gene transfer was followed by selection with colchicine, which selects for expression of the MDR1 gene, or with G418, which selects for expression of the neo gene. Thus, we could determine the function of the tricistronic vectors under conditions of selection for either the 5'-most or the 3'-most gene. In DNA-mediated transfections, we were able to achieve expression of all three open reading frames under either selection condition. We obtained higher expression of all three genes when colchicine was used to select for MDR1 expression than when G418 was used to select for neo expression. Expression of the non-selected GFP gene (the middle cistron) was unstable, most likely due to loss of integrated GFP DNA sequences during long-term culturing. We were able to achieve retrovirus-mediated transduction of all three genes, but this was an inefficient process.

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