OLIG gene targeting in human pluripotent stem cells for motor neuron and oligodendrocyte differentiation
OLIG gene targeting in human pluripotent stem cells for motor neuron and oligodendrocyte differentiation
复制标题
DOI：
10.1038/nprot.2011.310
复制
发表时间：
2011-05-01
期刊：
影响因子：
14.8
通讯作者：
Deng, Wenbin
中科院分区：
文献类型：
--
作者：
Liu, Ying;Jiang, Peng;Deng, Wenbin
多能干细胞可以进行基因标记以促进分化研究。在本文中，我们描述了一种基因靶向方案，将 GFP 盒敲入人多能干细胞 (hPSC) 的关键基因位点，然后使用基因标记的 hPSC 指导体外分化、免疫细胞化学和电生理学分析以及细胞移植后的体内表征。 Olig 转录因子在运动神经元 (MN) 和少突胶质细胞 (OL) 发生的转录调控途径中发挥关键作用。我们已经生成了 OLIG2-GFP hPSC 报告系，可以可靠地标记 MN 和 OL，以监测它们与 hPSC 的连续分化。 GFP 报告基因的表达概括了 OLIG 基因的内源表达。荧光激活细胞分选纯化细胞的体外表征与 MN 或 OL 谱系的细胞一致，具体取决于它们收集的阶段。该协议高效可靠，通常需要 5-7 个月才能完成。本文使用的遗传标签分化方法为将 hPSC 分化为其他谱系的类似工作提供了一般框架。
Pluripotent stem cells can be genetically labeled to facilitate differentiation studies. In this paper, we describe a gene-targeting protocol to knock in a GFP cassette into key gene loci in human pluripotent stem cells (hPSCs), and then use the genetically tagged hPSCs to guide in vitro differentiation, immunocytochemical and electrophysiological profiling and in vivo characterization after cell transplantation. The Olig transcription factors have key roles in the transcription regulatory pathways for the genesis of motor neurons (MNs) and oligodendrocytes (OLs). We have generated OLIG2-GFP hPSC reporter lines that reliably mark MNs and OLs for monitoring their sequential differentiation from hPSCs. The expression of the GFP reporter recapitulates the endogenous expression of OLIG genes. The in vitro characterization of fluorescence-activated cell sorting-purified cells is consistent with cells of the MN or OL lineages, depending on the stages at which they are collected. This protocol is efficient and reliable and usually takes 5-7 months to complete. The genetic tagging-differentiation methodology used herein provides a general framework for similar work for differentiation of hPSCs into other lineages.