1. Ashlee Heinecke Kimmel Lab SPUR 2008

2. Operationcleft.org

3. Wildtypeedn1 Mutantplcβ3 Mutant A D

4. Edn1 Plcβ3 Cell Proliferation/death Cell movement Jaw Morphology Goal: Clone a full length copy of plcβ3.

5. 28hpf 36hpf ~220 bp mRNA rRNA Extracted RNA Extract RNA Synthesize cDNA PCR RNAcDNA plcβ3 ~3.7kb Full length plcβ3 product Viable cDNA

6. plcβ3 Xba1 5’ 3’ EcoR1FLAG Tag Capturing full length plcβ3. EcoR1FLAG Tag plcβ3 Xba1 PCR Product:

7. Plasmid Vector plcβ3 Reformed Plasmid + Insert PCS2+ Vector ~4kb 3.7kb AATT TTAAGATC CTAG plcβ3 product

8. Reformed Plasmid + plcβ3 E. coli Transform AmpR +AMP 1.Grow bacteria from plate, generating more copies of the plasmid. 2.Extract plasmid DNA from bacteria. 3.Send off for sequencing.

9. 1.Use the full length clone of plcβ3 to create RNA that we can use to prefrom the rescue experiment to test the hypothesis. 2.Use FLAG-tag to visualize localization of Plcβ3 to further analyze its role in jaw development.

10. Acknowledgements Dr. O’Day and SPUR Mark Sasaki Chuck Kimmel Brian Eames Bonnie Ullmann April DeLaurier Allison Kunerth Jared Talbot And all other Kimmel kids. John Dowd