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Circ Res. 2012 Dec 7;111(12):1494-503. doi: 10.1161/CIRCRESAHA.112.274969. Epub 2012 Sep 11.

Genome editing of human embryonic stem cells and induced pluripotent stem cells with zinc finger nucleases for cellular imaging.

Author information

1
Department of Medicine, Division of Cardiology, Stanford School of Medicine, Stanford, CA, USA.

Abstract

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.

PMID:
22967807
PMCID:
PMC3518748
DOI:
10.1161/CIRCRESAHA.112.274969
[Indexed for MEDLINE]
Free PMC Article
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