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Craniosynostotic Rabbit Polymerase Chain Reaction-Suppression Subtractive Hybridization (PCR SSH)
James Cray Jr., Ph.D.1, Phillip Gallo, Ph.D.2, Emily Lensie, B.A.1, Joseph E. Losee, M.D.1, Mark P. Mooney, Ph.D.1,2,3, Sandeep Kathju, M.D. ,Ph.D.2,5, and Gregory M. Cooper, Ph.D.1,4,6
1Department
Pediatric Craniofacial Biology Laboratory
Department of Surgery
University of Pittsburgh
Divi
2010-03-29
Presenter: Dr. James Cray Jr., Ph.D.
Affidavit:
Director Name:
Author Category: Resident/Fellow
Presentation Category: Basic Science Research
Abstract Category: Craniomaxillofacial
In the United States, the incidence of craniosynostosis (CS) (premature fusion of the sutures of the cranial vault) is 1 in 2,000-3,000 live births. The condition has a range of severity from subclinical phenotypes to severe cases that exhibit increased intracranial pressure, severely altered head shape, mental retardation, and premature death. There exists a colony of rabbits with familial coronal craniosynostosis. This model has been instrumental in describing the craniometrics and growth and development after post-surgical therapy associated with craniosynostosis. The molecular analysis of this model is limited by the lack of molecular tools for use in rabbits. This project describes a novel methodology that compared differences in gene expression between normal (WT) and craniosynostotic rabbit tissues by polymerase chain reaction suppression subtractive hybridization (PCR SSH). Tissue was derived from the calvaria of 10-day old CS and WT rabbits. PCR SSH was performed to obtain cDNA clones enriched in either WT (underexpressed in CS mutant tissue) or CS (overexpressed) tissues. Fifteen candidate genes were identified as differentially expressed; using either pre-made rabbit real time PCR (Taqman) probes (Applied Biosystems) or custom designed rabbit probes (Applied Biosystems), 4 genes were confirmed by RT-PCR as being overexpressed in three CS pooled RNA samples: â-hemoglobin, Osteopontin (SPP1), SPARC, and CTSK. 2 genes were confirmed to be underexpressed in the CS pooled samples: COL3A1 and RNF12. This was an important first step in describing the molecular pathways affected within this novel CS model.
