Singh Amar M
Department of Biochemistry and Molecular Biology, Center for Molecular Medicine, University of Georgia, Athens, GA, United States.
Front Cell Dev Biol. 2019 Jan 31;7:11. doi: 10.3389/fcell.2019.00011. eCollection 2019.
Genomic manipulation of human pluripotent stem cells (hPSCs) has become essential to introduce genetic modifications and transgenes, and develop reporter lines. One of the major bottlenecks in gene editing is at the stage of single-cell cloning, which is thought to be variable across hPSC lines and is substantially reduced following a transfection. Due to the difficulty of performing fluorescent-assisted cell sorting (FACS) for single-cell isolation of hPSCs, previous approaches rely on manual colony picking, which is both time-consuming and labor-intensive. In this protocol, I provide a method for utilizing FACS to generate single-cell clones of hPSCs with efficiencies approaching 40% within 7-10 days. This can be achieved by sorting cells onto a feeder layer of MEFs in a stem cell defined medium with KSR and a Rock inhibitor, as early as 1-2 days following a transfection, streamlining the gene editing process. The approach described here provides a fundamental method for all researchers utilizing hPSCs for scientific studies.
对人类多能干细胞（hPSC）进行基因组操作已成为引入基因修饰和转基因以及开发报告系的关键。基因编辑的主要瓶颈之一在于单细胞克隆阶段，该阶段在不同的hPSC系中被认为存在差异，并且在转染后会大幅减少。由于对hPSC进行单细胞分离时难以进行荧光辅助细胞分选（FACS），以往的方法依赖于人工挑取集落，这既耗时又费力。在本方案中，我提供了一种利用FACS在7至10天内高效生成hPSC单细胞克隆的方法，效率接近40%。这可以通过在转染后1至2天，将细胞分选到含有KSR和一种Rho相关卷曲螺旋蛋白激酶（Rock）抑制剂的干细胞限定培养基中的小鼠胚胎成纤维细胞（MEF）饲养层上实现，从而简化基因编辑过程。这里描述的方法为所有利用hPSC进行科学研究的研究人员提供了一种基本方法。
