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...
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Published in | Cell reports (Cambridge) Vol. 23; no. 11; pp. 3146 - 3151 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
United States
Elsevier Inc
12.06.2018
Elsevier |
Subjects | |
Online Access | Get full text |
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Summary: | 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 forelimb 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.
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•Putative Tbx5 forelimb enhancers were deleted in mouse and zebrafish using CRISPR/Cas9•Double knockouts of the two enhancers show both are nonessential and nonredundant•A retinoic acid response element in one enhancer is nonessential for forelimb growth•Knockout studies are needed to infer necessity of proposed transcriptional enhancers
Forelimb development requires Tbx5. Using CRISPR/Cas9 gene editing to create homozygous deletions, Cunningham et al. show that two Tbx5 forelimb enhancers identified with reporter transgenes are not required for Tbx5 activation or forelimb development. These observations demonstrate that knockout studies are required to identify endogenous enhancers necessary for biological processes. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Lead Contact |
ISSN: | 2211-1247 2211-1247 |
DOI: | 10.1016/j.celrep.2018.05.052 |