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Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation
Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MyJove Corporation
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4781717/ https://www.ncbi.nlm.nih.gov/pubmed/26863600 http://dx.doi.org/10.3791/53583 |
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author | Blair, John D. Bateup, Helen S. Hockemeyer, Dirk F. |
author_facet | Blair, John D. Bateup, Helen S. Hockemeyer, Dirk F. |
author_sort | Blair, John D. |
collection | PubMed |
description | Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell's endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent stem cells. |
format | Online Article Text |
id | pubmed-4781717 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | MyJove Corporation |
record_format | MEDLINE/PubMed |
spelling | pubmed-47817172016-03-09 Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation Blair, John D. Bateup, Helen S. Hockemeyer, Dirk F. J Vis Exp Developmental Biology Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell's endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent stem cells. MyJove Corporation 2016-02-02 /pmc/articles/PMC4781717/ /pubmed/26863600 http://dx.doi.org/10.3791/53583 Text en Copyright © 2016, Journal of Visualized Experiments http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visithttp://creativecommons.org/licenses/by-nc-nd/3.0/ |
spellingShingle | Developmental Biology Blair, John D. Bateup, Helen S. Hockemeyer, Dirk F. Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation |
title | Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation |
title_full | Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation |
title_fullStr | Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation |
title_full_unstemmed | Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation |
title_short | Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation |
title_sort | establishment of genome-edited human pluripotent stem cell lines: from targeting to isolation |
topic | Developmental Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4781717/ https://www.ncbi.nlm.nih.gov/pubmed/26863600 http://dx.doi.org/10.3791/53583 |
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