The SKI Stem Cell Research Facility, The Center for Stem Cell Biology and Developmental Biology Program, Sloan Kettering Institute, 1275 York Avenue, New York, NY 10065, USA.
These authors contributed equally.
STAR Protoc. 2020 Sep 18;1(2). doi: 10.1016/j.xpro.2020.100052. Epub 2020 Jun 13.
Reporter cell lines based on human pluripotent stem cells (hPSCs) are highly desirable for studying differentiation, lineage tracing, and target cell selection. However, several technical bottlenecks, such as DNA transduction, low homology recombination rate (HDR), and single-cell cloning, have made this effort an arduous process in hPSCs. Here, we provide a step-by-step protocol and practical guide for generating reporter lines in hPSCs via CRISPR/Cas9-mediated HDR. We also elaborate on the process of generating a TBXT-GFP reporter line as an example.
基于人多能干细胞（hPSC）的报道细胞系对于研究分化、谱系追踪和靶细胞选择非常理想。然而，DNA 转导、低同源重组率（HDR）和单细胞克隆等几个技术瓶颈使得这一工作在 hPSC 中变得非常艰难。在这里，我们提供了一种通过 CRISPR/Cas9 介导的 HDR 在 hPSC 中生成报告基因系的分步方案和实用指南。我们还详细介绍了生成 TBXT-GFP 报告基因系的过程作为示例。
