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Investigating MSX1 Gene Implications in Orofacial Clefting Using Zebrafish Expression Models

This project aims to elucidate the role of the MSX1 gene in orofacial clefting, the most common congenital facial abnormality. Previous association studies have linked MSX1 to clefting in humans. Through bioinformatic analysis, two conserved enhancer regions (distal and proximal) were identified. The project focuses on creating a transgenic zebrafish model using the Tol2Kit and Gateway Cloning System to facilitate in vivo assays for testing DNA and protein-coding variants at the MSX1 locus. Acknowledgements include contributions from Dr. Peter Jezewski and others.

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Investigating MSX1 Gene Implications in Orofacial Clefting Using Zebrafish Expression Models

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  1. Zebrafish msxe Expression Analysis Cassie Wells

  2. Outline • msxe/MSX1 implication in orofacial clefting • Bioinformatic Analysis • My project goal • Tol2Kit / Gateway Cloning System • Expression Clone Design • Acknowledgements

  3. Cleft Lip and/or Palate • The most common congenital facial abnormality • Association studies have linked the gene MSX1 to clefting in humans • Murine Msx1 expression is controlled at the tips of the growing facial prominences by the two highly conserved distal and proximal enhancers, (DE and PE, respectively) Murine Msx1 expression is controlled at the tips of the growing facial prominences by the two highly conserved distal and proximal enhancers, (DE and PE, respectively) (MacKenzie et al., 1997)

  4. Bioinformatic Analysis Schematic diagram of the msxe locus • Bioinformatic analysis revealed two highly conserved enhancer regions (DE and PE), presumably corresponding to the murine enhancers identified by Mackenzie, and a putative promoter

  5. Conservation of the DE and Protein Binding Sites

  6. Project Purpose • My project purpose: To develop the first step, transgenic expression component, of an in vivo functional assay for DNA and protein coding variants at the MSX1 locus.

  7. 3 Fragment Recombination • Three PCR Products flanked by specific att sites and three Donor vectors are used in separate BP recombination reactions to generate three entry clones • The three entry clones and a destination vector are used together in a LR recombination reaction to create one expression clone

  8. pME B1. EGFP B2. msxe B3. msxe/MSX1 knock-out and rescue B4. MSX1 B5. mutant MSX1 Expression Clone Construct Possibilities p5E A1. Promoter A2. DE-Promoter A3. PE-Promoter A4. 2.1kb chunk (DE – Prom) p3E C1. pA C2. EGFPpA B pDestTol2pA2 A C

  9. Acknowledgements • Dr. Peter Jezewski (PI) • Divakar Prakash • Henry Jackson • Dr. Heidi Erlandsen • Institute of Oral Health Research

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