%0 Journal Article
%T Functional Assessment of Disease-Associated Regulatory Variants In Vivo Using a Versatile Dual Colour Transgenesis Strategy in Zebrafish
%A Shipra Bhatia&nbsp
%A Christopher T. Gordon&nbsp
%A Robert G. Foster&nbsp
%A Lucie Melin&nbsp
%A V¨¦ronique Abadie&nbsp
%A Genevi¨¨ve Baujat&nbsp
%A Marie-Paule Vazquez&nbsp
%A Jeanne Amiel&nbsp
%A Stanislas Lyonnet&nbsp
%A Veronica van Heyningen
%J PLOS Genetics
%D 2015
%I Public Library of Science (PLoS)
%R 10.1371/journal.pgen.1005193
%X Disruption of gene regulation by sequence variation in non-coding regions of the genome is now recognised as a significant cause of human disease and disease susceptibility. Sequence variants in cis-regulatory elements (CREs), the primary determinants of spatio-temporal gene regulation, can alter transcription factor binding sites. While technological advances have led to easy identification of disease-associated CRE variants, robust methods for discerning functional CRE variants from background variation are lacking. Here we describe an efficient dual-colour reporter transgenesis approach in zebrafish, simultaneously allowing detailed in vivo comparison of spatio-temporal differences in regulatory activity between putative CRE variants and assessment of altered transcription factor binding potential of the variant. We validate the method on known disease-associated elements regulating SHH, PAX6 and IRF6 and subsequently characterise novel, ultra-long-range SOX9 enhancers implicated in the craniofacial abnormality Pierre Robin Sequence. The method provides a highly cost-effective, fast and robust approach for simultaneously unravelling in a single assay whether, where and when in embryonic development a disease-associated CRE-variant is affecting its regulatory function.
%U http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.1005193