Isolation and partial sequencing of potentially odontoblast-specific/enriched rat cDNA clones obtained by suppression subtractive hybridization

Abstract

Odontoblasts, which are responsible for dentine formation, are known to synthesize unique gene products such as dentine sialophosphoprotein. To further identify and clone novel odontoblast-specific genes, a suppression subtractive hybridization technique was used here. Differentially or predominantly expressed cDNAs in odontoblasts of rat incisors were obtained by subtracting the common cDNAs expressed in odontoblasts, osteoblasts and pulp cells. Clones were then partially sequenced and analysed for nucleotide sequence homology by the basic local alignment search tool program. From a total of 1290 clones analysed, 538 odontoblast-enriched clones were identified in the subtracted cDNA library. Out of 538 clones, 498 clones (92.6%) demonstrated high identity with genes in the GenBank database. In contrast, 31 clones (5.7%) showed low sequence identity with known genes, among which 18 clones (3.3%) were observed more than once, thereby possibly representing odontoblast-specific/enriched genes. The majority (390 clones; 72.5%) of the clones with high homology to known genes were found to be the rat/mouse dentine sialophosphate by dot-blot analysis (326 clones) and sequencing (64 clones). The second highest enrichment (39 clones) was for phosphate-regulating gene with homology to endopeptidase on the X-chromosome, which codes for a neutral endopeptidase. After suppression subtractive hybridization, several cDNAs that are commonly present in osteoblasts and odontoblasts appeared unsuppressed. Therefore, a rat odontoblast-specific/enriched subtraction cDNA library has been created from which a number of potentially novel genes for odontoblasts could be identified.