一种高效的胚胎干细胞转染方法
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文章作者介绍了一种基于脂质体的高效siRNA转染方法。利用该protocol，可以在CCE细胞达到98%的转染效率，在D3细胞达到80%的转染效率，并且对干细胞的形态没有影响。
An efficient transfection method for mouse embryonic stem cells
B S Ko1,2,5, T C Chang1,5, S K Shyue3, Y C Chen4 and J Y Liou1
1National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan
2Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
3Institutes of Biomedical Science, Academic Sinica, Taipei, Taiwan
4Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan
Abstract
Embryonic stem （ES） cells are considered to have potentials for tissue regeneration and treatment of diverse human diseases. ES cells are capable of indefinite renewal and proliferation, which can be induced to differentiate into tissues of all three germ lines. Despite these exciting potential, it remains unclear as to how the renewal and differentiation programs are operated and regulated at the genetic level. Genetic manipulation such as delivery of exogenous gene expression or knockdown with small interfering RNA （siRNA） is commonly used in most of cancer or transformed cells but relatively rare in ES cells. In this study, we compare the transfection efficacies of several liposome-based transfection methods by introduction of a plasmid encoding enhanced green fluorescent protein （EGFP） into mouse ES （mES） cells. Our results show that transfection by Effectene achieves the efficiency of >98% in CCE and >80% in D3 cells. The optimal ratio of DNA:Effectene for EGFP transfection is between 1:4 and 1:8. Transient-expressed EGFP or endogenous protein kinase A （PKA） were significantly knocked down by Effectene transfection of specific siRNA. High EGFP level expression and accumulation in mES cells induces minor cytotoxicity but can be reduced by introducing siRNA of EGFP. Further, this transfection method did not significantly affect mES properties of proliferation or differentiation. Our results provide an optimal protocol to achieve an efficient transfection for mES cells.
Keywords: mouse embryonic stem cells, transfection, efficiency, green fluorescent protein, small interfering RNA
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