|ZFIN ID: ZDB-PUB-180614-19|
Genomic Knockout of Two Presumed Forelimb Tbx5 Enhancers Reveals They Are Nonessential for Limb Development
Cunningham, T.J., Lancman, J.J., Berenguer, M., Dong, P.D.S., Duester, G.
|Source:||Cell Reports 23: 3146-3151 (Journal)|
|Registered Authors:||Dong, P. Duc, Lancman, Joseph|
|Keywords:||CRISPR/Cas9 gene editing, Tbx5, enhancer, limb development, mouse, retinoic acid response element, zebrafish|
|PubMed:||29898387 Full text @ Cell Rep.|
Cunningham, T.J., Lancman, J.J., Berenguer, M., Dong, P.D.S., Duester, G. (2018) Genomic Knockout of Two Presumed Forelimb Tbx5 Enhancers Reveals They Are Nonessential for Limb Development. Cell Reports. 23:3146-3151.
ABSTRACTA 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.