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Functional Analysis of irf6 in Zebrafish via Designer Zinc Finger Nuclease Gene Targeting and Dominant Negative Transgenesis.
George N. Kamel, M.D., Graham Hickey, B.S., Max Dougherty, B.A., Michael Grimaldi, B.A., Renee Ethier Daigle, B.S., Eric C. Liao, M.D., Ph.D..
Massachusetts General Hospital, Boston, MA, USA.
Purpose: IRF6 is a transcription factor important for epithelial morphogenesis. Human mutation of IRF6 results in Van der Woude and popliteal pterygium syndromes. Mouse knockout of Irf6 results in cleft palate and constricted digits. Recently, IRF6 is confirmed as a key locus for non-syndromic cleft lip and palate from large population genome-wide association studies. Meanwhile, the zebrafish has been shown to be an excellent vertebrate model to assess cleft phenotypes. We aim to study the molecular and morphogenetic mechanism of IRF6 function in vertebrate craniofacial development. We are carrying out targeted disruption of irf6 using a novel designer zinc finger nuclease (ZFN) approach, to assess the loss-of-function phenotype. We are also generating dominant negative mutants to study the gain-of-function phenotype. We believe these complementary studies will provide additional insight into the mechanism of irf6 function during palatal development.
Methods: Dominant-negative irf6 mutant genes were created based on known human mutations, using site-directed mutagenesis at arginine 84 (R84H, R84C, R84L) and a truncation mutant (Irf6ΔCT) carrying only the sequence for the protein-binding domain, ablating the DNA binding domain. These irf6 mutants are then placed under the transcriptional control of either the heat-shock (hsp) or sox10 promoter, thereby conferring inducibility or neural crest specificity. ZFN targeting irf6 were engineered using a modular assembly approach. Using ZiFit web based software, irf6 zinc finger nuclease target sites were identified. Three-finger arrays were then assembled for each zinc finger DNA binding domain, and were selected for optimal binding affinity to their target sites using a bacterial two-hybrid system. These irf6 targeting zinc finger nucleases are then transcribed in vitro and injected into fertilized embryos to mediate the specific DNA breaks at the irf6 locus, creating heritable gene-specific mutations.
Results: Eight irf6 transgenic lines were produced, hsp: Irf6R84H, R84C, R84L and Irf6ΔCT and sox10: Irf6R84H, R84C, R84L and Irf6ΔCT. The hsp lines all demonstrate induction with heat shock treatment. The sox10 lines demonstrate ectopic expression of irf6 in the sox10 spatiotemporal pattern. We have also successfully performed the bacterial two-hybrid screen for the irf6 targeting ZFN arrays (Fig. 1). We are now in the process of evaluating the phenotype of the irf6 dominant-negative and loss-of-function mutants.
Conclusion: irf6 plays an essential role for vertebrate palate development. Our study would explore irf6 function in the craniofacial development of zebrafish, enable us to better extrapolate zebrafish phenotypes to higher vertebrates. Additionally, the complementary gain and loss-of function genetic analysis would further elucidate the mechanism of irf6 function in vertebrate palatogenesis.
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