A platform for rapid generation of single and multiplexed reporters in human iPSC lines

Authors:
Pei Y, Sierra G, Sivapatham R, Swistowski A, Rao MS, Zeng X
In:
Source: Scientific Reports
Publication Date: (2015)
Issue: 5: 9205
Research Area:
Gene Expression
Cells used in publication:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
Platform:
Nucleofector™ I/II/2b
Experiment
Reporter iPSC lines generation Prior to nucleofection, XCL1 iPSC were maintained and passed using Accutase (Life Tech., NJ) to make sure cells are growing in monolayer. On the day of nucleofection, single cell suspension cells were generated using Accutase followed by inactivation and washes with HBSS. 4–6 µg of each pair of TALENs/ZFNs RNA was used for nucleofection using Amaxa Human Stem Cell Nucleofection Kit (Lonza, NJ). After nucleofection, cells were plated in mTeSR™1 medium with 10 µM Rock inhibitor. After 2–5 days recovery, cells were treated with appropriate antibiotics. Drug resistant colonies were re-plated at low density for single cell cloning. Colonies growing from single cells were screened by PCRs and sequencing to identify targets with correct donor vector integrations. The verified targets were expanded, stored and characterized for future experiments.
Abstract
Induced pluripotent stem cells (iPSC) are important tools for drug discovery assays and toxicology screens. In this manuscript, we design high efficiency TALEN and ZFN to target two safe harbor sites on chromosome 13 and 19 in a widely available and well-characterized integration-free iPSC line. We show that these sites can be targeted in multiple iPSC lines to generate reporter systems while retaining pluripotent characteristics. We extend this concept to making lineage reporters using a C-terminal targeting strategy to endogenous genes that express in a lineage-specific fashion. Furthermore, we demonstrate that we can develop a master cell line strategy and then use a Cre-recombinase induced cassette exchange strategy to rapidly exchange reporter cassettes to develop new reporter lines in the same isogenic background at high efficiency. Equally important we show that this recombination strategy allows targeting at progenitor cell stages, further increasing the utility of the platform system. The results in concert provide a novel platform for rapidly developing custom single or dual reporter systems for screening assays.