Zebrafish msxe Expression AnalysisCassie Wells

Outline

0msxe/MSX1 implication in orofacial clefting0Bioinformatic Analysis0My project goal0Tol2Kit / Gateway Cloning System0Expression Clone Design 0Acknowledgements

Cleft Lip and/or Palate0 The most common congenital facial

abnormality0 Association studies have linked the

gene MSX1 to clefting in humans0 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)

Bioinformatic Analysis

Schematic diagram of the msxe locus

0 Bioinformatic analysis revealed two highly conserved enhancer regions (DE and PE), presumably corresponding to the murine enhancers identified by Mackenzie, and a putative promoter

Conservation of the DE and Protein Binding Sites

Project Purpose

0My 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.

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

pDestTol2pA2

A

B

C

p5E

A1. Pro

moter

A2. DE-

Prom

oter

A3. PE-

Prom

oter

A4. 2.1kb

chunk

(DE – Pro

m)

pMEB1. EGFPB2. msxeB3. msxe/MSX1 knock-out and rescueB4. MSX1B5. mutant MSX1

p3E

C1. pA

C2. EGFPpA

Expression Clone Construct

Possibilities

Acknowledgements

0Dr. Peter Jezewski (PI)0Divakar Prakash0Henry Jackson0Dr. Heidi Erlandsen0 Institute of Oral Health Research