Institute of Biomedicine and Biotechnology of Cantabria (IBBTEC), CSIC/Universidad de Cantabria, Albert Einstein 22, 39011 Santander, Spain; Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Campus-Vienna-Biocenter 1, 1030 Vienna, Austria.
Institute of Biomedicine and Biotechnology of Cantabria (IBBTEC), CSIC/Universidad de Cantabria, Albert Einstein 22, 39011 Santander, Spain.
STAR Protoc. 2022 Jun 21;3(3):101492. doi: 10.1016/j.xpro.2022.101492. eCollection 2022 Sep 16.
-regulatory elements (CREs) orchestrate the spatiotemporal control of gene expression. The regulatory activity of CREs is typically assessed by reporter assays, in which CREs are studied outside their endogenous context. To circumvent this problem, we developed a CRISPR-Cas9 knockin approach to study CREs in a scar-free genomic context. Here, we describe the design, transfection, and screening protocol to insert CREs in mouse embryonic stem cells. Our strategy can provide important insights into the sufficiency of CREs for gene expression control. For complete details on the use and execution of this protocol, please refer to Pachano et al. (2021).
-调控元件（CREs）协调基因表达的时空控制。通常通过报告基因检测来评估 CRE 的调控活性，在这种检测中，CRE 是在其内源环境之外进行研究的。为了避免这个问题，我们开发了一种 CRISPR-Cas9 基因敲入方法，以便在无疤痕的基因组环境中研究 CRE。在这里，我们描述了在小鼠胚胎干细胞中插入 CRE 的设计、转染和筛选方案。我们的策略可以为 CRE 对基因表达控制的充分性提供重要的见解。有关该方案的使用和执行的完整详细信息，请参阅 Pachano 等人（2021 年）。
