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, Carla B Mellough, Birthe Dorgau, Stefan Przyborski, Inmaculada Moreno-Gimeno, Majlinda Lako

Research output: Contribution to journalArticle

11 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)311-321
Number of pages11
JournalStem Cells
Volume34
Issue number2
DOIs
Publication statusPublished - Feb 2016
Externally publishedYes

Fingerprint

Pluripotent Stem Cells
Vertebrate Photoreceptor Cells
Homeobox Genes
Zinc Fingers
Green Fluorescent Proteins
Cell Differentiation
Technology
Clone Cells
Embryoid Bodies
Cell Line
Puromycin
Retinal Ganglion Cells
Genomic Instability
Homozygote
3' Untranslated Regions
Human Embryonic Stem Cells
Reverse Transcriptase Polymerase Chain Reaction
Flow Cytometry
Transcription Factors
Epithelium

Cite this

@article{13f046d013b444e8b81931aa03ef9c3d,
title = "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",
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 = "3' Untranslated Regions, Cell Differentiation, Cell Line, Genes, Reporter, Green Fluorescent Proteins, Homeodomain Proteins, Human Embryonic Stem Cells, Humans, Photoreceptor Cells, Ribonucleases, Trans-Activators, Zinc Fingers, Journal Article, Research Support, Non-U.S. Gov't",
author = "Joseph Collin and Mellough, {Carla B} and Birthe Dorgau and Stefan Przyborski and Inmaculada Moreno-Gimeno and Majlinda Lako",
note = "{\circledC} 2015 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.",
year = "2016",
month = "2",
doi = "10.1002/stem.2240",
language = "English",
volume = "34",
pages = "311--321",
journal = "Stem Cells",
issn = "1066-5099",
publisher = "Alphamed Press",
number = "2",

}

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, Joseph; Mellough, Carla B; Dorgau, Birthe; Przyborski, Stefan; Moreno-Gimeno, Inmaculada; Lako, Majlinda.

In: Stem Cells, Vol. 34, No. 2, 02.2016, p. 311-321.

Research output: Contribution to journalArticle

TY - JOUR

T1 - 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

AU - Collin, Joseph

AU - Mellough, Carla B

AU - Dorgau, Birthe

AU - Przyborski, Stefan

AU - Moreno-Gimeno, Inmaculada

AU - Lako, Majlinda

N1 - © 2015 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

PY - 2016/2

Y1 - 2016/2

N2 - 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.

AB - 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.

KW - 3' Untranslated Regions

KW - Cell Differentiation

KW - Cell Line

KW - Genes, Reporter

KW - Green Fluorescent Proteins

KW - Homeodomain Proteins

KW - Human Embryonic Stem Cells

KW - Humans

KW - Photoreceptor Cells

KW - Ribonucleases

KW - Trans-Activators

KW - Zinc Fingers

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1002/stem.2240

DO - 10.1002/stem.2240

M3 - Article

VL - 34

SP - 311

EP - 321

JO - Stem Cells

JF - Stem Cells

SN - 1066-5099

IS - 2

ER -