Cloning Of Cbfa-1 CDNA Fragment From Rat Dental Papilla Cell And Investigation Of Cbfa1 Expression In Rat Dental Papilla

Core-binding factor a l( cbfal), also called osteoblast-specific transcription factor 2 (OSF2), is a recently found transcription factor that relates closely to bone development. Cbfal is considered to be a member of runt family because it comprises a DNA binding domain that is highly homologous to runt domain of drosophila category. Core-binding factor alpha 1 (Cbfal) plays an essential role in bone formation and osteoblast differentiation and serves as a master gene regulating osteoblast-specific gene expression. The gene is expressed in cells of the osteoblast lineage only, and when expressed in non-skeletal cells, the cells assume many of the characteristics of an osteoblast. In knockout experiments, no evidence of bone formation could be observed in animals that are homozygous for the deletion. Studies of the heterozygote indicate that osteoblast function is highly compromised, including a severe reduction in osteoblastic cell number, bone matrix protein synthesis and the activity of alkaline phosphatase. All studies above show a very important role cbfal plays in bone formation and thus makes it now possible to deepen our understanding of the molecular mechanism of bone formation as well as osteoblast differentiation. As mineralized tissues, tooth and bone share many similarities in hard tissue formation. Current studies have been focused on osteoblast differentiation and bone development while little attention is paid to tooth development. In this study, we investigated the gene expression pattern of Cbfal in odontoblast as well as tooth development by gene cloning and in situ hybridization. The results revealed the essential role cbfal plays in tooth development. Simultaneously, we also generated Cbfal cDNA probe and anti-sense Cbfal eukaryotic expression system which, as part of the series study, lie basis for further investigation. 1. Cloning and sequencing of Cbfal cDNA fragment Cbfal gene transfection to odontoblasts plays vital part in comprehensive functional study of Cbfal. However, whether Cbfal is expressed in odontoblast is yet unclear. In this study, Cbfal cDNA fragment was successfully cloned from primary cultures of rat papilla cell by RT-PCR method, demonstrating that Cbfal is expressed in this cell. Meanwhile, this study also makes preparations for in situ hybridization and anti-sense Cbfal transfection. 2. Cbfal cDNA probe preparation and its expression in rat tooth germ As hard tissue, tooth and bone share many similarities in development processes. Previous studies have demonstrated that Cbfal expression appears at the early stage of bone development and decreases as soon as bone development is finished. This indicates that Cbfal takes part in bone development process. Whether this gene expression conservation also happens in tooth development is not clear. In the present experiment, we generated dig-labeled Cbfal cDNA probe and performed in situ hybridization in rat tooth germs of different stages. The results indicated that cbfal is expressed in papilla cells and odontoblasts of early stage tooth germs, namely cap stage and early bell stage. The expression is down-regulated in late bell stage of the above-mentioned two cell types but not ameloblasts. Cbfal participates tooth germ development and like HOX genes, appears in sites of fast development activities and soon decreases after developmental process. Interestingly, Cbfal...