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Generation of GFP report lines using home-made AAVS1-specific TALENs in hiPSCs

Friday, November 08, 2013 — Poster Session III

10:00 a.m. – 12:00 p.m.

FAES Academic Center (Upper-Level Terrace)




  • Y Luo
  • T Cerbini
  • MS Rao
  • J Zou


Generation of reporter line in hiPSCs provides enormous potentials in both basic stem cell research and regenerative medicine. AAVS1 (adeno-associated virus integration site 1) locus is a “safe harbor” and has an open chromatin structure with insulator elements that maintain expression of transgene. Here, using a Godden Gate Assembly Kit, we have designed and made AAVS1-specific TALENs (Transcription Activator-like Effector Nucleases), a new class of artificial restriction enzymes that has been shown to significantly promote homologous recombination by > 1000 fold. Using this site-specific TALEN, we have successfully created two hiPSC reporter lines in both ND2 and NL5 hiPSCs. Their constitutive expressions of green fluorescent protein (GFP) at AAVS1 locus were maintained not only at undifferentiated stage but also in spontaneous differentiating toward three germ lines. Moreover, we have found that this sustained GFP expression occurred only in the presence of CMV early enhancer/chicken b actin (CAG) promoter, but not by EF1a promoter in AAVS1 site. Our data suggest that a TALEN-mediated gene targeting is a valuable tool for studies of lineage development, gene function and correction in hiPSCs.

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