Open AccessGeneration of GFP Reporter Human Induced Pluripotent Stem Cells Using AAVS1 Safe Harbor Transcription Activator‐Like Effector Nuclease
Author(s) -
Luo Yongquan,
Rao Mahendra,
Zou Jizhong
Publication year - 2014
Publication title -
current protocols in stem cell biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.658
H-Index - 28
eISSN - 1938-8969
pISSN - 1941-7322
DOI - 10.1002/9780470151808.sc05a07s29
Subject(s) - transcription activator like effector nuclease , biology , microbiology and biotechnology , effector , nuclease , induced pluripotent stem cell , activator (genetics) , green fluorescent protein , stem cell , transcription factor , embryonic stem cell , genome editing , gene , genetics , crispr
Generation of a fluorescent GFP reporter line in human induced pluripotent stem cells (hiPSCs) provides enormous potentials in both basic stem cell research and regenerative medicine. A protocol for efficiently generating such an engineered reporter line by gene targeting is highly desired. Transcription activator‐like effector nucleases (TALENs) are a new class of artificial restriction enzymes that have been shown to significantly promote homologous recombination by >1000‐fold. The AAVS1 (adeno‐associated virus integration site 1) locus is a “safe harbor” and has an open chromatin structure that allows insertion and stable expression of transgene. Here, we describe a step‐by‐step protocol from determination of TALENs activity, hiPSC culture, and delivery of a donor into AAVS1 targeting site, to validation of targeted integration by PCR and Southern blot analysis using hiPSC line, and a pair of open‐source AAVS1 TALENs. Curr. Protoc. Stem Cell Biol . 29:5A.7.1‐5A.7.18. © 2014 by John Wiley & Sons, Inc.