Chatterjee Papri, Cheung Yuri, Liew Chee
UCR Stem Cell Center, Department of Cell Biology and Neuroscience, University of California Riverside, USA.
J Vis Exp. 2011 Oct 5(56):3110. doi: 10.3791/3110.
Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs). While improvements in several gene delivery methods have been described, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.
基因修饰仍然是研究干细胞生物学以及阐述人类胚胎干细胞（HESC）潜在临床应用的重要工具。虽然已经描述了几种基因递送方法的改进，但转染对于HESC来说仍然是一个变幻莫测的过程，并且在人类诱导多能干细胞（iPSC）中尚未见报道。在本视频中，我们展示了我们实验室如何使用带有增强型绿色荧光蛋白（eGFP）报告基因的质粒对人类iPSC进行常规转染和核转染。人类iPSC经过适应性培养并维持无饲养层培养，以消除饲养层细胞转染的可能性，并允许在转染后有效选择稳定的转基因iPSC克隆。对于核转染，人类iPSC先用ROCK抑制剂预处理，用胰蛋白酶消化成小细胞团，进行核转染，然后接种到饲养层细胞条件培养基中的饲养层上，以提高细胞回收率。6小时后可获得表达转基因的人类iPSC。24小时后进行抗生素筛选，1周内出现稳定的转基因株系。我们的方案对于人类iPSC系来说是稳健且可重复的，同时不会改变这些细胞的多能性。
