Department of Hematology and Immunology, Myeloma Center Brussels, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium.
J Biotechnol. 2010 Oct 1;150(1):37-40. doi: 10.1016/j.jbiotec.2010.07.010. Epub 2010 Jul 16.
The development of SIN-lentivectors has paved the way for the double copy vectors (DCV), which substitute the deletion in the 3'LTR with either transgenic or insulator sequences. However, the limits of this approach remain unclear. Previous results have demonstrated that transduction efficiencies of DCV carrying large insulator inserts in their 3'LTRs were impaired in a size-dependent manner. We wondered if this was also true for promoter-transgene inserts and whether they remained functional upon integration into the genome. Therefore, we designed a series of DCV with increasing 3'LTR sizes containing different promoter-transgene combinations. Transduced cells were scored for both transduction efficiency and insert functionality. We found that the transduction efficiencies indeed were impaired in a size-dependent way. Efficiency with inserts below 1 kb linearly decreased with size, while sizes between 1 and 2 kb showed a further decrease to a minimum of 5% of an original "empty" SIN-vector. However, we did not find an LTR size that completely abolished transduction. Moreover, we demonstrated that all inserts remained functional regardless the promoter-transgene combination used. Therefore, we conclude from our data that DCV indeed remain functional, but transduction efficiencies drop radically when inserts larger than 1 kb are being used.
SIN 载体的发展为双拷贝载体 (DCV) 铺平了道路，后者用转基因或绝缘子序列替代了 3'LTR 中的缺失。然而，这种方法的局限性尚不清楚。先前的结果表明，在其 3'LTR 中携带大的绝缘子插入物的 DCV 的转导效率以大小依赖的方式受损。我们想知道这对于启动子-转基因插入物是否也成立，以及它们在整合到基因组中后是否仍然具有功能。因此，我们设计了一系列具有不同启动子-转基因组合的 3'LTR 大小增加的 DCV。转导细胞的转导效率和插入物功能都进行了评分。我们发现，转导效率确实以大小依赖的方式受损。插入物小于 1 kb 的效率呈线性下降，而大小在 1 到 2 kb 之间的效率进一步下降到原始“空”SIN 载体的 5%以下。然而，我们没有发现完全消除转导的 LTR 大小。此外，我们证明无论使用何种启动子-转基因组合，所有插入物都保持功能。因此，我们从数据中得出结论，DCV 确实仍然具有功能，但当使用大于 1 kb 的插入物时，转导效率会急剧下降。
