Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System

Shenglan Li; Haipeng Xue; Bo Long; Li Sun; Tai Truong; Ying Liu

doi:10.3791/52539

的hiPSC神经细胞系特异性敲开记者代高效使用CRISPR / Cas9和Cas9双切口酶系统

JoVE Journal
Developmental Biology

A subscription to JoVE is required to view this content. Sign in or start your free trial.

JoVE Journal Developmental Biology

Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System

的hiPSC神经细胞系特异性敲开记者代高效使用CRISPR / Cas9和Cas9双切口酶系统

Full Text

11,717 Views

14:46 min

May 28, 2015

DOI: 10.3791/52539-v

Shenglan Li*^1,2, Haipeng Xue*^1,2, Bo Long^1,2,5, Li Sun^1,2,6, Tai Truong^1,2,4,7, Ying Liu^1,2,3

¹Department of Neurosurgery,The University of Texas Health Science Center at Houston, ²Center for Stem Cell and Regenerative Medicine, Brown Foundation Institute of Molecular Medicine,The University of Texas Health Science Center at Houston, ³The Senator Lloyd & B. A. Bentsen Center for Stroke Research, Brown Foundation Institute of Molecular Medicine,The University of Texas Health Science Center at Houston, ⁴Summer Research Program, Office of Educational Programs,The University of Texas Health Science Center at Houston, ⁵Department of Anesthesiology,Shengjing Hospital, China Medical University, ⁶Department of Oncology, Renji Hospital,Shanghai Jiaotong University School of Medicine, ⁷Biology Department,University of West Georgia

Please note that some of the translations on this page are AI generated. Click here for the English version.

基因组编辑工具，如 CRISPR（成簇规则间隔短回文重复序列）/Cas（CRISPR 相关）系统，大大提高了人类诱导多能干细胞（hiPSC）中的基因靶向效率。本手稿描述了使用 CRISPR/Cas 系统辅助同源重组生成谱系特异性 hiPSC 报告基因的方案。

该程序的总体目标是使用 CRISPR Cas 9 介导的同源重组生成谱系特异性人诱导多能干细胞或 H-I-P-S-C 敲入报告基因。这是通过首先设计和构建目标向量来实现的。第二步是为 CRISPR Cas 9 系统或 CAS 9 N 双缺口病例系统设计和构建单向导 RNA。

2 93 英尺。然后用单向导 RNA 和 CAS 9 表达载体对细胞进行 cot 转染，提取并扩增 DNA，以便通过 T 7 核酸内切酶 one 测定进行评估。最后一步是对潜在的脱靶位点进行计算机模拟预测。网站。最终，HI PSC 将用靶向载体和 CRISPR Cas 九系统组分转染，以制备神经谱系敲入报告细胞系。

View the full transcript and gain access to thousands of scientific videos