Quantitative assessment on the cloning efficiencies of lentiviral transfer vectors with a unique clone site.
Quantitative assessment on the cloning efficiencies of lentiviral transfer vectors with a unique clone site.
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DOI：
10.1038/srep00415
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发表时间：
2012
影响因子：
4.6
通讯作者：
Tandon, Anurag
中科院分区：
文献类型：
--
作者：
Zhang, Gang;Tandon, Anurag
慢病毒载体（LV）是体内和体外转基因表达的有力工具。然而，LV的构建是低效率的，由于大的尺寸和缺乏合适的克隆位点。因此，开发有效的策略来克隆LV至关重要。在这里，我们报告了一种组合策略，以有效地构建LV使用绿色荧光蛋白，hPlk 2野生型（WT）和突变体基因作为插入。首先，进行定点诱变（SDM）以产生插入片段的BamH I位点;其次，在BamH I消化后使pWPI LV去磷酸化;最后，优化插入片段和载体DNA的量和比例以增加单体连接。结果表明，阳性克隆的总百分率约为48%± 7.6%。使用这种方法，几乎所有的载体都可以通过两个或三个minipreps构建。因此，我们的研究提供了一个有效的方法来构建大规模的载体。
Lentiviral vectors (LVs) are powerful tools for transgene expression in vivo and in vitro. However, the construction of LVs is of low efficiency, due to the large sizes and lack of proper clone sites. Therefore, it is critical to develop efficient strategies for cloning LVs. Here, we reported a combinatorial strategy to efficiently construct LVs using EGFP, hPlk2 wild type (WT) and mutant genes as inserts. Firstly, site-directed mutagenesis (SDM) was performed to create BamH I site for the inserts; secondly, pWPI LV was dephosphorylated after BamH I digestion; finally, the amounts and ratios of the insert and vector DNA were optimized to increase monomeric ligation. Our results showed that the total percentage of positive clones was approximately 48%±7.6%. Using this method, almost all the vectors could be constructed through two or three minipreps. Therefore, our study provided an efficient method for constructing large-size vectors.
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