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.; Mellough, C.B.; Dorgau, B.; Przyborski, S.; Moreno-Gimeno, I.; Lako, M.

doi:10.1002/stem.2240

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.; Mellough, C.B.; Dorgau, B.; Przyborski, S.; Moreno-Gimeno, I.; Lako, M.

Authors

J. Collin

C.B. Mellough

B. Dorgau

Professor Stefan Przyborski stefan.przyborski@durham.ac.uk
Executive Dean

I. Moreno-Gimeno

M. Lako

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.

Citation

Collin, J., Mellough, C., Dorgau, B., Przyborski, S., Moreno-Gimeno, I., & 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), 311-321. https://doi.org/10.1002/stem.2240

Journal Article Type	Article
Acceptance Date	Oct 11, 2015
Online Publication Date	Nov 26, 2015
Publication Date	Feb 1, 2016
Deposit Date	Jan 4, 2016
Publicly Available Date	Jan 18, 2016
Journal	STEM CELLS
Print ISSN	1066-5099
Electronic ISSN	1549-4918
Publisher	Oxford University Press
Peer Reviewed	Peer Reviewed
Volume	34
Issue	2
Pages	311-321
DOI	https://doi.org/10.1002/stem.2240
Keywords	Zinc finger nucleases, Cone-rod homeobox, Human embryonic stem cells, Photoreceptor precursors.

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© 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.