Generation of GFP Reporter Human Induced Pluripotent Stem Cells Using AAVS1 Safe Harbor Transcription Activator-Like Effector Nuclease.
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.
NIH Center for Regenerative Medicine, Laboratory of Stem Cell Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Medicine, Bethesda, Maryland.
Yongquan
Y
Luo
Mahendra
M
Rao
Jizhong
J
Zou
D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D000229:Dependovirus; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D016662:Virus Integration
D049452:Green Fluorescent Proteins; D003851:Deoxyribonucleases
adeno-associated virus integration site 1 (AAVS1); gene targeting; induced pluripotent stem cells (iPSCs); reporter cell line; transcription activator-like effector nucleases (TALENs)