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Using zinc finger nuclease technology to generate CRX-reporter human embryonic stem cells as a tool to identify and study the emergence of photoreceptors precursors during pluripotent stem cell differentiation.

Collin, J. and Mellough, C.B. and Dorgau, B. and Przyborski, S. and Moreno-Gimeno, I. and Lako, M. (2016) 'Using zinc finger nuclease technology to generate CRX-reporter human embryonic stem cells as a tool to identify and study the emergence of photoreceptors precursors during pluripotent stem cell differentiation.', Stem cells., 34 (2). pp. 311-321.

Abstract

The purpose of this study was to generate human embryonic stem cell (hESC) lines harboring the green fluorescent protein (GFP) reporter at the endogenous loci of the Cone-Rod Homeobox (CRX) gene, a key transcription factor in retinal development. Zinc finger nucleases (ZFNs) designed to cleave in the 3′ UTR of CRX were transfected into hESCs along with a donor construct containing homology to the target region, eGFP reporter, and a puromycin selection cassette. Following selection, polymerase chain reaction (PCR) and sequencing analysis of antibiotic resistant clones indicated targeted integration of the reporter cassette at the 3′ of the CRX gene, generating a CRX-GFP fusion. Further analysis of a clone exhibiting homozygote integration of the GFP reporter was conducted suggesting genomic stability was preserved and no other copies of the targeting cassette were inserted elsewhere within the genome. This clone was selected for differentiation towards the retinal lineage. Immunocytochemistry of sections obtained from embryoid bodies and quantitative reverse transcriptase PCR of GFP positive and negative subpopulations purified by fluorescence activated cell sorting during the differentiation indicated a significant correlation between GFP and endogenous CRX expression. Furthermore, GFP expression was found in photoreceptor precursors emerging during hESC differentiation, but not in the retinal pigmented epithelium, retinal ganglion cells, or neurons of the developing inner nuclear layer. Together our data demonstrate the successful application of ZFN technology to generate CRX-GFP labeled hESC lines, which can be used to study and isolate photoreceptor precursors during hESC differentiation.

Item Type:Article
Keywords:Zinc finger nucleases, Cone-rod homeobox, Human embryonic stem cells, Photoreceptor precursors.
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Status:Peer-reviewed
Publisher Web site:http://dx.doi.org/10.1002/stem.2240
Publisher statement:© 2015 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Date accepted:11 October 2015
Date deposited:18 January 2016
Date of first online publication:26 November 2015
Date first made open access:No date available

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