Genome Editing of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells With Zinc Finger Nucleases for Cellular ImagingNovelty and Significance
Rationale: Molecular imaging has proven to be a vital tool in the characterization of stem cell behavior in vivo. However, the integration of reporter genes has typically relied on random integration, a method that is associated with unwanted insertional mutagenesis and positional effects on transgene expression.
Objective: To address this barrier, we used genome editing with zinc finger nuclease (ZFN) technology to integrate reporter genes into a safe harbor gene locus (PPP1R12C, also known as AAVS1) in the genome of human embryonic stem cells and human induced pluripotent stem cells for molecular imaging.
Methods and Results: We used ZFN technology to integrate a construct containing monomeric red fluorescent protein, firefly luciferase, and herpes simplex virus thymidine kinase reporter genes driven by a constitutive ubiquitin promoter into a safe harbor locus for fluorescence imaging, bioluminescence imaging, and positron emission tomography imaging, respectively. High efficiency of ZFN-mediated targeted integration was achieved in both human embryonic stem cells and induced pluripotent stem cells. ZFN-edited cells maintained both pluripotency and long-term reporter gene expression. Functionally, we successfully tracked the survival of ZFN-edited human embryonic stem cells and their differentiated cardiomyocytes and endothelial cells in murine models, demonstrating the use of ZFN-edited cells for preclinical studies in regenerative medicine.
Conclusion: Our study demonstrates a novel application of ZFN technology to the targeted genetic engineering of human pluripotent stem cells and their progeny for molecular imaging in vitro and in vivo.
- homologous recombination
- induced pluripotent stem cells
- molecular imaging
- reporter gene
- zinc finger nuclease
- Received June 2, 2012.
- Revision received August 29, 2012.
- Accepted September 11, 2012.
- © 2012 American Heart Association, Inc.