Xiong Kai, Zhou Yan, Hyttel Poul, Bolund Lars, Freude Kristine Karla, Luo Yonglun
Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Grønnegårdsvej 7, 1870 Frederiksberg C, Denmark.
Danish Regenerative Engineering Alliance for Medicine (DREAM), Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 4, 8000, Aarhus C, Denmark.
Stem Cell Res. 2016 Nov;17(3):665-669. doi: 10.1016/j.scr.2016.10.011. Epub 2016 Nov 17.
Human fibroblasts were engineered to express the CRISPR-based synergistic activation mediator (SAM) complex: dCas9-VP64 and MS2-P65-HSF1. Two induced pluripotent stem cells (iPSCs) clones expressing SAM were established by transducing these fibroblasts with lentivirus expressing OCT4, SOX2, KLF4 and C-MYC. We have validated that the reprogramming cassette is silenced in the SAM iPSC clones. Expression of pluripotency genes (OCT4, SOX2, LIN28A, NANOG, GDF3, SSEA4, and TRA-1-60), differentiation potential to all three germ layers, and normal karyotypes are validated. These SAM-iPSCs provide a novel, useful tool to investigate genetic regulation of stem cell proliferation and differentiation through CRISPR-mediated activation of endogenous genes.
人类成纤维细胞经基因工程改造后表达基于CRISPR的协同激活介质（SAM）复合物：dCas9-VP64和MS2-P65-HSF1。通过用表达OCT4、SOX2、KLF4和C-MYC的慢病毒转导这些成纤维细胞，建立了两个表达SAM的诱导多能干细胞（iPSC）克隆。我们已经验证了重编程盒在SAM iPSC克隆中是沉默的。多能性基因（OCT4、SOX2、LIN28A、NANOG、GDF3、SSEA4和TRA-1-60）的表达、向所有三个胚层的分化潜能以及正常核型均得到验证。这些SAM-iPSC提供了一种新颖且有用的工具，可通过CRISPR介导的内源性基因激活来研究干细胞增殖和分化的遗传调控。
