Liou Jun-Yang, Ko Bor-Sheng, Chang Tzu-Ching
Institute of Cellular and System Medicine, National Health Research Institutes, 35, Keyan Road, Zhunan, Miaoli County, 35053, Taiwan.
Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan.
Methods Mol Biol. 2017;1622:139-147. doi: 10.1007/978-1-4939-7108-4_11.
Embryonic stem (ES) cells are an important source of stem cells in tissue engineering and regenerative medicine because of their high self-renewal capacities and differentiation potentials. However, the detailed molecular mechanisms controlling the differentiation and renewal programs in ES cells remained unclear. One of the difficulties in understanding these mechanisms substantially results from the low efficacies of gene manipulation by delivering exogenous gene expression or knockdown of endogenous gene expression with small interfering RNA (siRNA) in ES cells. Here we describe an optimized protocol for efficiently transfecting mouse ES cells by Effectene, a liposome-based method. The high transfection efficiency in mouse ES cells is demonstrated in this chapter by (1) achieving a percentage of enhanced green fluorescence protein (EGFP) expression in >98% embryoid bodies after introducing plasmids encoding the protein; (2) decreased SOX-2 and Oct-3/4 expression and subsequent morphological evidences of cell differentiation after introducing siRNA expression for suppressing SOX-2 and Oct-3/4, which are known to be essential for maintenance of stem cell properties in mouse ES cells; and (3) overexpression or attenuated expression of 14-3-3σ to regulate cell proliferation of mouse ES cells.
胚胎干细胞（ES细胞）因其高自我更新能力和分化潜能，成为组织工程和再生医学中干细胞的重要来源。然而，控制ES细胞分化和更新程序的详细分子机制仍不清楚。深入理解这些机制的困难之一，很大程度上源于在ES细胞中通过递送外源基因表达或用小干扰RNA（siRNA）敲低内源性基因表达来进行基因操作的效率较低。在此，我们描述了一种通过基于脂质体的Effectene方法高效转染小鼠ES细胞的优化方案。本章通过以下方式证明了在小鼠ES细胞中的高转染效率：（1）在导入编码增强型绿色荧光蛋白（EGFP）的质粒后，>98%的胚状体中实现了EGFP表达；（2）在导入用于抑制SOX-2和Oct-3/4的siRNA表达后，SOX-2和Oct-3/4表达降低，随后出现细胞分化的形态学证据，已知SOX-2和Oct-3/4对维持小鼠ES细胞的干细胞特性至关重要；（3）14-3-3σ的过表达或表达减弱以调节小鼠ES细胞的增殖。
