Exit from Pluripotency Assay of Mouse Embryonic Stem Cells
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Author:
Date:
2017-08-20
[Abstract] A novel method to assess the dissolution of the core pluripotency transcription-factor circuit of mouse Embryonic Stem Cells (mESCs) has been developed (Ying et al., 2003; Betschinger et al., 2013). In order to efficiently identify genes essential for the break-down of the pluripotency network in mutant mESCs with proliferation defects, we adapted this ‘exit from pluripotency assay’ (Bodak et al., 2017; Cirera-Salinas et al., 2017). The protocol described here has been successfully applied to several mESC lines and is easily transposable from one laboratory to another.
[摘要] 已经开发了评估小鼠胚胎干细胞(mESCs)的核心多能转录因子电路溶解的新方法(Ying等,2003; Betschinger等,2013)。 为了有效识别具有增殖缺陷的突变体mESCs中多能网络分解所必需的基因,我们调整了这种“多能性测定法”(Bodak等,2017; Cirera-Salinas等,2017)。 这里描述的方案已经成功应用于几个mESC系列,并且可以容易地从一个实验室转座到另一个实验室。 【背景】几十年来,科学家已经尝试确定基因与一般(例如胚胎体)或定向(例如,神经元前体细胞)分化方案的mESCs的分化潜能的机制。最近,发现2i培养基允许在体外俘获天真的干细胞(Ying et al。,2008)。 ...
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Targeted Nucleotide Substitution in Mammalian Cell by Target-AID
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Author:
Date:
2017-06-05
[Abstract] Programmable RNA-guided nucleases based on CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) systems have been applied to various type of cells as powerful genome editing tools. By using activation-induced cytidine deaminase (AID) in place of the nuclease activity of the CRISPR/Cas9 system, we have developed a genome editing tool for targeted nucleotide substitution (C to T or G to A) without donor DNA template (Figure 1; Nishida et al., 2016). Here we describe the detailed method for Target-AID to perform programmable point mutagenesis in the genome of mammalian cells. A specific method for targeting the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in Chinese Hamster Ovary (CHO) cell was described here as an ...
[摘要] 基于CRISPR的可编程RNA引导核酸酶(集群定期交织的短回文重复)-Cas(CRISPR相关蛋白)系统已被应用于各种类型的细胞作为强大的基因组编辑工具。通过使用激活诱导的胞苷脱氨酶(AID)代替CRISPR / Cas9系统的核酸酶活性,我们开发了一种用于靶向核苷酸替代(C至T或G至A)的基因组编辑工具,无供体DNA模板(图1 ; Nishida等人,2016)。这里我们描述Target-AID在哺乳动物细胞基因组中进行可编程点突变的详细方法。在这里描述了用于靶向中国仓鼠卵巢(CHO)细胞中的次黄嘌呤 - 鸟嘌呤磷酸核糖基转移酶(HPRT)基因的具体方法作为实例,而该方法主要应适用于任何感兴趣的基因广泛的细胞类型。
图1. Target-AID及其可靶向位点的示意图。在指导RNA(gRNA)依赖性方式中,通过接头与nCas9(D10A)融合的PmCDA1在-21周围进行可编程胞苷突变至相对于哺乳动物细胞中非互补链上的PAM序列的-16位。可目标地点是根据以前的工作中观察到的有效的基础替代(> 20%)来确定的。 ...
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