Department of Dermatology, University of Colorado School of Medicine, Aurora, CO, USA.
Charles C. Gates Center for Regenerative Medicine, University of Colorado School of Medicine, Aurora, CO, USA.
Methods Mol Biol. 2020;2155:11-21. doi: 10.1007/978-1-0716-0655-1_2.
The discovery of induced pluripotent stem cell (iPSC) technology has provided a versatile platform for basic science research and regenerative medicine. With the rise of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) systems and the ease at which they can be utilized for gene editing, creating genetically modified iPSCs has never been more advantageous for studying both organism development and potential clinical applications. However, to better understand the behavior and true therapeutic potential of iPSCs and iPSC-derived cells, a tool for labeling and monitoring these cells in vitro and in vivo is needed. Here, we describe a protocol that provides a straightforward method for introducing a stable, highly expressed fluorescent protein into iPSCs using the CRISPR/Cas9 system and a standardized donor vector. The approach involves the integration of the EGFP transgene into the transcriptionally active adeno-associated virus integration site 1 (AAVS1) locus through homology directed repair. The knockin of this transgene results in the generation of iPSC lines with constitutive expression of the EGFP protein that also persists in differentiated iPSCs. These EGFP-labeled iPSC lines are ideal for assessing iPSC differentiation in vitro and evaluating the distribution of iPSC-derived cells in vivo after transplantation into model animals.
诱导多能干细胞（iPSC）技术的发现为基础科学研究和再生医学提供了一个多功能平台。随着规律成簇间隔短回文重复（CRISPR）/CRISPR 相关蛋白 9（Cas9）系统的兴起，以及它们在基因编辑中的易用性，创建基因修饰的 iPSC 从未如此有利于研究生物发育和潜在的临床应用。然而，为了更好地理解 iPSC 和 iPSC 衍生细胞的行为和真正的治疗潜力，需要一种用于在体外和体内标记和监测这些细胞的工具。在这里，我们描述了一种使用 CRISPR/Cas9 系统和标准化供体载体将稳定、高表达荧光蛋白引入 iPSC 的简单方法。该方法涉及通过同源定向修复将 EGFP 转基因整合到转录活跃的腺相关病毒整合位点 1（AAVS1）位点。该转基因的敲入导致产生持续表达 EGFP 蛋白的 iPSC 系，并且在分化的 iPSC 中也持续存在。这些 EGFP 标记的 iPSC 系非常适合评估体外 iPSC 分化，并评估移植到模型动物后 iPSC 衍生细胞的分布。
