October 9 - 12, 2012
Building 10 & Natcher Conference Center
- General Schedule of Events
- Opening Plenary Session
- Concurrent Symposia Sessions
- Poster Sessions
- National Graduate Student Research Conference
- FARE Award Ceremony
- Special Exhibits on Resources for Intramural Research
- TSA Research Festival Exhibit Show
- Clinical Center Tours
- Research Festival Committees
- Past Research Festivals
Efficient Multiplex Safe Harbor Gene-Targeting and Recombinase-Mediated Cassette Exchange in Human iPSCs
| Friday, November 08, 2013 — Poster Session III | |||
|---|---|---|---|
10:00 a.m. – 12:00 p.m. |
FAES Academic Center (Upper-Level Terrace) |
NIAMS |
STEMCELL-1 |
Authors
- T Cerbini
- Y Luo
- M Rao
- J Zou
Abstract
Unlocking the potential of Induced Pluripotent Stem Cells (iPSCs) will require genome engineering to enable the optimization of differentiation protocols and the tracking of transplanted cells in vivo. To this end, we have generated multiple human iPSC lines by gene tareting at two defined loci that allow constitutive expression of the inserted reporter genes. The loci utilized are so called “safe harbors” in the genome which, due to their open chromatin structure, allow transcription of the introduced transgenes robustly and persistently. These iPSC lines contain lox-flanked CAG driven fluorescent protein genes at the well-defined AAVS1 locus in the PPP1R12C (protein-phosphatase 1 regulatory subunit 12C) on Chr. 19 and a new safe harbor, C13, in intron 2 of the CLYBL (citrate lyase beta-like) on Chr. 13. Cell lines retained their pluripotency after gene-targeting, as assayed by staining for pluripotency markers and by in vitro spontaneous differentiation via embryoid body formation. We show that flow cytometry enabled quantification of the relative expression conferred by the number of transgene integrations, and allowed the quick identification of clones containing single-allele, double-allele, and random integrations. Recombinase-mediated cassette exchange functionality was confirmed in both AAVS1 targeted and AAVS1/C13 double-targeted human iPSC lines.
