UNIT 5A.7 Generation of GFP Reporter Human Induced Pluripotent Stem Cells Using AAVS1 Safe Harbor Transcription Activator-Like Effector Nuclease
Published Online: 16 MAY 2014
Copyright © 2013 John Wiley & Sons, Inc. All rights reserved.
Lab Protocol Title
Current Protocols in Stem Cell Biology
How to Cite
Luo, Y., Rao, M. and Zou, J. 2014. Generation of GFP Reporter Human Induced Pluripotent Stem Cells Using AAVS1 Safe Harbor Transcription Activator-Like Effector Nuclease. Current Protocols in Stem Cell Biology. 29:A:5A.7:5A.7.1–5A.7.18.
- Published Online: 16 MAY 2014
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.
- gene targeting;
- transcription activator-like effector nucleases (TALENs);
- adeno-associated virus integration site 1 (AAVS1);
- induced pluripotent stem cells (iPSCs);
- reporter cell line