PUBLICATION

Functional Assessment of Disease-Associated Regulatory Variants In Vivo Using a Versatile Dual Colour Transgenesis Strategy in Zebrafish

Authors
Bhatia, S., Gordon, C.T., Foster, R.G., Melin, L., Abadie, V., Baujat, G., Vazquez, M.P., Amiel, J., Lyonnet, S., Heyningen, V.V., Kleinjan, D.A.
ID
ZDB-PUB-150602-2
Date
2015
Source
PLoS Genetics   11: e1005193 (Journal)
Registered Authors
Bhatia, Shipra, Gordon, Chris, Lyonnet, Stanislas
Keywords
Zebrafish, Embryos, Hypothalamus, Morpholino, Gene expression, Transcription factors, Enhancer elements, Point mutation
MeSH Terms
  • Repressor Proteins/genetics
  • Repressor Proteins/metabolism
  • Zebrafish Proteins/genetics
  • Zebrafish Proteins/metabolism
  • Green Fluorescent Proteins/genetics
  • Green Fluorescent Proteins/metabolism
  • Eye Proteins/genetics
  • Eye Proteins/metabolism
  • Homeodomain Proteins/genetics
  • Homeodomain Proteins/metabolism
  • Interferon Regulatory Factors/genetics
  • Interferon Regulatory Factors/metabolism
  • Regulatory Elements, Transcriptional*
  • Pierre Robin Syndrome/genetics*
  • Transgenes*
  • Paired Box Transcription Factors/genetics
  • Paired Box Transcription Factors/metabolism
  • Hedgehog Proteins/genetics
  • Hedgehog Proteins/metabolism
  • PAX6 Transcription Factor
  • Animals
  • Protein Binding
  • Zebrafish
(all 23)
PubMed
26030420 Full text @ PLoS Genet.
Abstract
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.
Genes / Markers
Figures
Figure Gallery (7 images)
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Expression
Phenotype
Mutations / Transgenics
Allele Construct Type Affected Genomic Region
zf994TgTransgenic Insertion
    zf995TgTransgenic Insertion
      zf996TgTransgenic Insertion
        zf997TgTransgenic Insertion
          zf998TgTransgenic Insertion
            zf999TgTransgenic Insertion
              zf1000TgTransgenic Insertion
                zf1001TgTransgenic Insertion
                  zf1002TgTransgenic Insertion
                    zf1003TgTransgenic Insertion
                      1 - 10 of 24
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                      Human Disease / Model
                      Human Disease Fish Conditions Evidence
                      Weissenbacher-Zweymuller syndromeTAS
                      1 - 1 of 1
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                      Sequence Targeting Reagents
                      Target Reagent Reagent Type
                      six3aMO1-six3a,six3bMRPHLNO
                      six3bMO1-six3a,six3bMRPHLNO
                      1 - 2 of 2
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                      Fish
                      Antibodies
                      Name Type Antigen Genes Isotypes Host Organism
                      Ab1-six3polyclonal
                        IgGRabbit
                        1 - 1 of 1
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                        Orthology
                        No data available
                        Engineered Foreign Genes
                        Marker Marker Type Name
                        EGFPEFGEGFP
                        mCherryEFGmCherry
                        1 - 2 of 2
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                        Mapping
                        No data available