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. 2016 Feb;34(2):311-21.
doi: 10.1002/stem.2240. Epub 2015 Nov 26.

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

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Free PMC article

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

Joseph Collin et al. Stem Cells. .
Free PMC article

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.

Keywords: Cone-rod homeobox; Human embryonic stem cells; Photoreceptor precursors; Zinc finger nucleases.

Figures

Figure 1
Figure 1
Zinc finger nuclease‐mediated targeting of green fluorescent protein (GFP) to the 3′ UTR of Cone‐rod homeobox (CRX). (A): Schematic diagram of endogenous CRX gene structure, targeting cassette, targeting strategy and sequencing data from sections of the insertion site, the endogenous CRX‐exogenous eGFP border site and 3′ homology arm‐endogenous CRX border, obtained from three analyzed human embryonic stem cell (hESC) clones; (B): Polymerase chain reaction (PCR) of three hESC clones with primers spanning the integration site to assess whether the cassette had integrated at the correct site. Depending on size of band produced (upper band of 5067 bp with integration, lower band of 2171 bp without) and whether the clone has a heterozygous integration (by the presence of the upper and lower bands), or a homozygote integration (a single upper band), such as clone 1 in the first lane. Lanes 2 and 3 show two clones with heterozygote integration, lanes 4–6 are genomic DNA from control untreated H9 hESCs and lane 7, a no template negative PCR control. This is a representative example of at least three repeats; (C): Quantitative PCR (qPCR)‐mediated copy number analysis indicating presence of two copies of GFP (and hence targeting cassette) in CRX‐GFP hESC clone 1(c1) and its absence from wild type H9 hESC genomic DNA, data are presented as the mean ± SEM (n = 3). Abbreviations: CRX, cone‐rod homeobox; GFP, green fluorescent protein; hESC, human embryonic stem cell.
Figure 2
Figure 2
Green fluorescent protein (GFP) reporter accurately mimics the expression of endogenous cone‐rod homeobox (CRX) during human embryonic stem cell (hESC) differentiation. (A–L): Immunocytochemistry with antibodies raised against CRX and GFP showing correlation at day 30 (A–D), day 60 (E–H), and day 90 (I–L) of hESC differentiation. (M): Detection of GFP without antibody using confocal microscopy. Scale bars = 20 µm. Abbreviations: CRX, Cone‐rod homeobox; GFP, green fluorescent protein.
Figure 3
Figure 3
Cone‐rod homeobox (CRX)/green fluorescent protein (GFP) expression is found in photoreceptor precursors at day 60 of human embryonic stem cell (hESC) differentiation. Immunocytochemistry with antibodies raised against GFP and Recoverin (A, B), OPN1SW (C), OPN1MW/LW (D), Calbindin (E), HuC/D (F), and Ki‐67 (G). Scale bars = 20 µm (A, F), 5 µm (B), and 10 µm (C, D, E). Abbreviation: GFP, green fluorescent protein
Figure 4
Figure 4
Cone‐rod homeobox (CRX) expression in photoreceptor precursors assessed by immunocytochemistry and quantitative real‐time polymerase chain reaction (qRT‐PCR) at day 90 of differentiation. Immunocytochemistry with antibodies raised against green fluorescent protein (GFP) and Recoverin (A), OPN1SW (B), OPN1MW/LW (C), PKCα (D), HuC/D (E), and Ki‐67 (F). Scale bars = 10 µm (E) and 20 µm (A, B, C, D, F); (G) Flow activated cell sorting of GFP‐positive (green) and GFP‐negative (red) populations; (H) and (I) qRT‐PCR analysis of unsorted, flow activated cell sorted GFP‐positive (Sorted GFP+) and GFP‐negative (Sorted GFP‐) populations and fetal retina at 18 weeks of gestation (18 weeks fetal retina), indicating CRX, RECOVERIN (RCVRN), and CONE ARRESTIN (C‐ARR) expression in the GFP‐positive population. Expression of short, medium, and long wave opsins (OPN1SW, OPN1MW, and OPN1LW) and rhodopsin (RHO) are also shown. Data are presented as the mean ± SEM (n = 3). Significant differences found are marked with an asterisk (*). Abbreviation: GFP, green fluorescent protein.

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