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

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

Current protocols in stem cell biology (2014-05-20)
Yongquan Luo, Mahendra Rao, Jizhong Zou
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.

MATERIALS
Product Number
Brand
Product Description

Roche
Anti-Digoxigenin-AP, Fab fragments, from sheep
Roche
DIG Wash and Block Buffer Set, storage temp.:2-8°C
Roche
DNA Molecular Weight Marker II, DIG-labeled, solution, pkg of 500 μL (10 μg/ml)
Roche
Nylon Membranes, positively charged, roll W × L 0.3 m × 3 m, sheet W × L 10 cm × 15 cm, sheet W × L 20 cm × 30 cm
Roche
DIG Easy Hyb Granules, powder, suitable for hybridization
Roche
PCR DIG Probe Synthesis Kit, sufficient for 25 reaction (50 μL final reaction volume)