Department of Genetics and Yale Stem Cell Center, Yale University, New Haven, CT, USA.
Biotechniques. 2019 Jan;66(1):23-27. doi: 10.2144/btn-2018-0115.
Gene regulatory networks that control pluripotency of human embryonic stem cells (hESCs) are of considerable interest for regenerative medicine. RNAi and CRISPR/Cas9 technologies have allowed the identification of hESC regulators on a genome-wide scale. However, these technologies are ill-suited for mechanistic studies because knockdown/knockout clones of essential genes do not grow in culture. We have developed a genetic rescue strategy that combines CRISPR/Cas9-mediated knockout with TALEN-mediated integration of a doxycycline-inducible rescue transgene into a constitutive AASV1 locus. The resulting rescue clones are stable in culture, allow modulation of the rescue transgene dosage by titration of doxycycline in the media and can be combined with various molecular assays, thus providing mechanistic insights into gene function in a variety of cellular contexts.
用于控制人类胚胎干细胞（hESC）多能性的基因调控网络对于再生医学具有重要意义。RNAi 和 CRISPR/Cas9 技术已允许在全基因组范围内鉴定 hESC 调节剂。然而，这些技术不适用于机制研究，因为必需基因的敲低/敲除克隆在培养中不能生长。我们开发了一种遗传挽救策略，该策略将 CRISPR/Cas9 介导的敲除与 TALEN 介导的将四环素诱导的挽救转基因整合到组成型 AASV1 基因座中结合起来。由此产生的挽救克隆在培养中稳定，允许通过在培养基中滴定四环素来调节挽救转基因的剂量，并且可以与各种分子测定相结合，从而在各种细胞环境中提供对基因功能的机制见解。
