Identification of a novel gene isolated from a fracture callus

We have previously hypothesized that a vast number of genes are expressed during the fracture repair. Past work in our laboratory identified many of these genes, involved specifically during fracture healing, some being functionally unknown (identified as ESTs (Expressed Sequence Tags) or as completely novel). One such EST was selected for further studies based on its expression pattern (from microarray experiments), its highest level of mRNA expression on post-fracture (PF) day 10 and 14. Since a partial sequence is known, a "contig" approach was utilized to construct the full-length gene. A final contig of 1686 by was constructed. Primers were designed to amplify 1121 by of the contig using RT-PCR. However, no band was obtained. Simultaneously, primers were designed for the amplification of the original EST which was obtained. This fragment was subcloned, sequenced, and its identity verified. Further, northern analysis was performed using RNA isolated from intact bone as well as PF day 3, 5, 7, 10, 14, and 21 callus. Results indicate this gene was differentially expressed, with high levels on PF days 7-21, and its mRNA size was ∼6500 bp. The task remains to obtain the full-length gene and characterize its structure and function.