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.
Highlights
Induced pluripotent stem cells are important tools for drug discovery assays and toxicology screens
We design high efficiency transcription activator-like effector nuclease (TALEN) and zinc finger nucleases (ZFN) to target two safe harbor sites on chromosome 13 and 19 in a widely available and well-characterized integration-free Induced pluripotent stem cells (iPSC) line. We show that these sites can be targeted in multiple iPSC lines to generate reporter systems while retaining pluripotent characteristics
We demonstrate that we can develop a master cell line strategy and 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
Summary
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. To further extend the utility of iPSC and somatic cells for such assays we designed high efficiency TALEN to target safe harbor sites on chromosomes 19 (Chr. 19) and 13 (Chr. 13) to generate monoallelic and biallelic reporters We further extended this targeting strategy to making lineage-specific reporters by targeting a Nanoluc-Halotag construct to the C-terminal of the GFAP and MAP2 genes. Overall our results confirm that we have a novel platform for rapidly developing single and multiplexed reporter lines, and the high efficiency constructs can be used to make similar lines in other normal and patient specific lines
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