不同转导方式及载体形式对TALENs在小鼠胚胎干细胞打靶效率的研究

Different Delivery Methods of TALENs Affect the Targeting Efficiency in Mouse Embryonic Stem Cells

通过同源重组进行小鼠胚胎干细胞基因敲除是目前应用最为广泛的基因敲除技术手段,但同源重组的效率却非常低.TALEs和FokI的剪切结构域融合的TALENs可以实现高效的基因敲除.已有报导表明,TALENs识别位点的DNA序列和长度,及间隔区的长短等条件影响TALENs打靶效率.但外源载体的导入方式及载体的形式(DNA或RNA)是否以及如何影响TALENs的打靶效率还不明确.用不同的转导方式将TALENs导入小鼠胚胎干细胞中,并检测TALENs切割识别位点的效率,实验发现Lipofectamine○R2000转染的TALENs比电转化能更高效地切割目的 DNA位点,而DNA质粒或体外转录的RNA表达的TALENs切割DNA位点的效率相近.数据表明,在应用TALENs进行基因敲除时,有必要选择合适的转导方式以提高突变效率.

Homologous recombination has been widely applied to knockout genes in mouse embryonic stem cells. But the efficiency of homologous recombination is very low. With the emerging TALENs technolo- gy, high efficiency of gene knqckout could be achieved. Many factors, such as the sequences and the length of TALENs recognition sites, and the length of the spacer, affect TALENs targeting efficiency. However, whether and how the delivery methods of TALENs contribute to TALENs targeting efficiency has not been in- vestigated. In this study, we compared the targeting efficiency of TALENs delivered by Lipofectamine~ 2000 transfection and by electroporation in mouse embryonic stem cells, and found that TALENs delivered by Lipo- fectamine 2000 cut the targeting site more efficiently. Moreover, TALENs expressed by DNA plasmids or in vitro transcribed RNAs cut DNA with similar efficiency. Our data suggested that appropriate delivery method of TALENs should be ehosen to achieve high targeting efficiency.