Duch Mogens, Carrasco Maria L, Jespersen Thomas, Hansen Bettina D, Pedersen Finn Skou
Department of Molecular Biology, University of Aarhus, DK8000, Aarhus, Denmark.
Gene. 2004 Mar 31;329:61-9. doi: 10.1016/j.gene.2003.12.032.
Retroviral vectors that are able to sustain multiple rounds of replication may find many applications. However, one critical feature of such vectors is the ability to maintain an intact transgene cassette during repeated rounds of replication. We here report on the stability of a translational cassette consisting of an internal ribosome entry site followed by the enhanced green fluorescent protein coding sequence inserted in different configurations into murine leukemia virus genomes. In two of the constructs, the insert was located in the upstream part of the U3 region while in the third construct it was inserted in the 3' untranslated region of the viral genome. Furthermore, in two of the constructs, the translational cassette was flanked by a direct repeat, while no such structure flanked the third construct. Our results show that deletion of the heterologous translational cassette is observed for all constructs upon extended cell culture and that the number of replication rounds before revertants are detected can be postponed by decreasing the length of the repeat flanking the translational cassette.
能够维持多轮复制的逆转录病毒载体可能有许多应用。然而，此类载体的一个关键特征是在重复复制轮次期间维持完整转基因盒的能力。我们在此报告一个翻译盒的稳定性，该翻译盒由一个内部核糖体进入位点和随后的增强型绿色荧光蛋白编码序列组成，以不同构型插入小鼠白血病病毒基因组。在其中两个构建体中，插入片段位于U3区域的上游部分，而在第三个构建体中，它插入到病毒基因组的3'非翻译区。此外，在其中两个构建体中，翻译盒两侧有一个直接重复序列，而第三个构建体两侧没有这种结构。我们的结果表明，在延长细胞培养后，所有构建体都观察到异源翻译盒的缺失，并且通过缩短翻译盒两侧重复序列的长度，可以推迟检测到回复突变体之前的复制轮数。
