Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Cunningham, Thomas J., Lancman, Joseph J., Berenguer, Marie, Dong, P. Duc Si, Duester, Gregg
Formato: Online Artículo Texto
Lenguaje:English
Publicado: 2018
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
_version_ 1783337920984252416
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
work_keys_str_mv AT cunninghamthomasj genomicknockoutoftwopresumedforelimbtbx5enhancersrevealstheyarenonessentialforlimbdevelopment
AT lancmanjosephj genomicknockoutoftwopresumedforelimbtbx5enhancersrevealstheyarenonessentialforlimbdevelopment
AT berenguermarie genomicknockoutoftwopresumedforelimbtbx5enhancersrevealstheyarenonessentialforlimbdevelopment
AT dongpducsi genomicknockoutoftwopresumedforelimbtbx5enhancersrevealstheyarenonessentialforlimbdevelopment
AT duestergregg genomicknockoutoftwopresumedforelimbtbx5enhancersrevealstheyarenonessentialforlimbdevelopment