Unit

UNIT 5A.7 Generation of GFP Reporter Human Induced Pluripotent Stem Cells Using AAVS1 Safe Harbor Transcription Activator-Like Effector Nuclease

  1. Yongquan Luo,
  2. Mahendra Rao,
  3. Jizhong Zou

Published Online: 16 MAY 2014

DOI: 10.1002/9780470151808.sc05a07s29

Current Protocols in Stem Cell Biology

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.

Author Information

  1. NIH Center for Regenerative Medicine, Laboratory of Stem Cell Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Medicine, Bethesda, Maryland

Publication History

  1. Published Online: 16 MAY 2014

Abstract

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.

Keywords:

  • gene targeting;
  • transcription activator-like effector nucleases (TALENs);
  • adeno-associated virus integration site 1 (AAVS1);
  • induced pluripotent stem cells (iPSCs);
  • reporter cell line