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Genomic Knockout of Two Presumed Forelimb Tbx5 Enhancers Reveals They Are Nonessential for Limb Development
A standard approach in the identification of transcriptional enhancers is the use of transgenic animals carrying DNA elements joined to reporter genes inserted randomly in the genome. We examined elements near Tbx5, a gene required for forelimb development in humans and other vertebrates. Previous t...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6034701/ https://www.ncbi.nlm.nih.gov/pubmed/29898387 http://dx.doi.org/10.1016/j.celrep.2018.05.052 |
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author | Cunningham, Thomas J. Lancman, Joseph J. Berenguer, Marie Dong, P. Duc Si Duester, Gregg |
author_facet | Cunningham, Thomas J. Lancman, Joseph J. Berenguer, Marie Dong, P. Duc Si Duester, Gregg |
author_sort | Cunningham, Thomas J. |
collection | PubMed |
description | A standard approach in the identification of transcriptional enhancers is the use of transgenic animals carrying DNA elements joined to reporter genes inserted randomly in the genome. We examined elements near Tbx5, a gene required for forelimb development in humans and other vertebrates. Previous transgenic studies reported a mammalian Tbx5 fore-limb enhancer located in intron 2 containing a putative retinoic acid response element and a zebrafish tbx5a forelimb (pectoral fin) enhancer located downstream that is conserved from fish to mammals. We used CRISPR/Cas9 gene editing to knockout the endogenous elements and unexpectedly found that deletion of the intron 2 and downstream elements, either singly or together in double knockouts, resulted in no effect on forelimb development. Our findings show that reporter transgenes may not identify endogenous enhancers and that in vivo genetic loss-of-function studies are required, such as CRISPR/Cas9, which is similar in effort to production of animals carrying reporter transgenes. |
format | Online Article Text |
id | pubmed-6034701 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
record_format | MEDLINE/PubMed |
spelling | pubmed-60347012018-07-06 Genomic Knockout of Two Presumed Forelimb Tbx5 Enhancers Reveals They Are Nonessential for Limb Development Cunningham, Thomas J. Lancman, Joseph J. Berenguer, Marie Dong, P. Duc Si Duester, Gregg Cell Rep Article A standard approach in the identification of transcriptional enhancers is the use of transgenic animals carrying DNA elements joined to reporter genes inserted randomly in the genome. We examined elements near Tbx5, a gene required for forelimb development in humans and other vertebrates. Previous transgenic studies reported a mammalian Tbx5 fore-limb enhancer located in intron 2 containing a putative retinoic acid response element and a zebrafish tbx5a forelimb (pectoral fin) enhancer located downstream that is conserved from fish to mammals. We used CRISPR/Cas9 gene editing to knockout the endogenous elements and unexpectedly found that deletion of the intron 2 and downstream elements, either singly or together in double knockouts, resulted in no effect on forelimb development. Our findings show that reporter transgenes may not identify endogenous enhancers and that in vivo genetic loss-of-function studies are required, such as CRISPR/Cas9, which is similar in effort to production of animals carrying reporter transgenes. 2018-06-12 /pmc/articles/PMC6034701/ /pubmed/29898387 http://dx.doi.org/10.1016/j.celrep.2018.05.052 Text en http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). |
spellingShingle | Article Cunningham, Thomas J. Lancman, Joseph J. Berenguer, Marie Dong, P. Duc Si Duester, Gregg Genomic Knockout of Two Presumed Forelimb Tbx5 Enhancers Reveals They Are Nonessential for Limb Development |
title | Genomic Knockout of Two Presumed Forelimb Tbx5 Enhancers Reveals They Are Nonessential for Limb Development |
title_full | Genomic Knockout of Two Presumed Forelimb Tbx5 Enhancers Reveals They Are Nonessential for Limb Development |
title_fullStr | Genomic Knockout of Two Presumed Forelimb Tbx5 Enhancers Reveals They Are Nonessential for Limb Development |
title_full_unstemmed | Genomic Knockout of Two Presumed Forelimb Tbx5 Enhancers Reveals They Are Nonessential for Limb Development |
title_short | Genomic Knockout of Two Presumed Forelimb Tbx5 Enhancers Reveals They Are Nonessential for Limb Development |
title_sort | genomic knockout of two presumed forelimb tbx5 enhancers reveals they are nonessential for limb development |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6034701/ https://www.ncbi.nlm.nih.gov/pubmed/29898387 http://dx.doi.org/10.1016/j.celrep.2018.05.052 |
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