生成 PAX7 报告细胞以研究人类多能干细胞的骨骼肌生成。

Generation of PAX7 Reporter Cells to Investigate Skeletal Myogenesis from Human Pluripotent Stem Cells.

机构信息

Department of Microbiology, Immunology, and Molecular Genetics, University of California Los Angeles, Los Angeles, CA 90095, USA.

Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California Los Angeles, Los Angeles, CA 90095, USA.

出版信息

STAR Protoc. 2020 Nov 5;1(3):100158. doi: 10.1016/j.xpro.2020.100158. eCollection 2020 Dec 18.

DOI:10.1016/j.xpro.2020.100158

PMID:33377052

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7757361/

Abstract

This protocol describes the use of CRISPR/Cas9-mediated homology-directed recombination to construct a PAX7-GFP reporter in human pluripotent stem cells (hPSCs). PAX7 is a key transcription factor and regulator of skeletal muscle stem/progenitor cells. We obtained heterozygous knockin reporter cells and validated their PAX7 expression using both artificial activation by the CRISPR/dCas9-VPR system and physiological activation during hPSC myogenic differentiation. These cells can serve as tools for better understanding of hPSC myogenesis and enriching myogenic cells for downstream analysis. For complete details on the use and execution of this protocol, please refer to Xi et al. (2017) and Xi et al. (2020).

摘要

本方案描述了利用 CRISPR/Cas9 介导的同源重组技术在人多能干细胞（hPSC）中构建 PAX7-GFP 报告基因的方法。PAX7 是骨骼肌干细胞/祖细胞的关键转录因子和调节因子。我们获得了杂合敲入报告细胞，并通过 CRISPR/dCas9-VPR 系统的人工激活和 hPSC 成肌分化过程中的生理激活验证了其 PAX7 的表达。这些细胞可作为更好地理解 hPSC 成肌作用以及富集肌细胞进行下游分析的工具。如需了解本方案的使用和实施的详细信息，请参考 Xi 等人（2017 年）和 Xi 等人（2020 年）的文献。