Search for key molecules in bone and cartilage differentiation

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Various type of signaling molecules and transcription factors are involved in bone and cartilage differentiation. Studies based on loss of function indicate that Runx 2, Osterix, and Sox 9 are indispensable for development of such hard tissues. Although those molecules are critical for skeletetogenesis, there still would be other uncharacterized molecules that govern bone and cartilage differentiation. To search for such molecules, we tried to establish a functional screening assay system specifically utilized for cartilage differentiation. We used type Xl collagen promoter as a monitor to differentiate into chondrocyte, since endogenous type Xl collagen expression is induced following the cartilage differentiation. We transfected collagen promoter plus beta-galactosidase construct (pXlcol-Z) into a teratocarcinoma derived fibroblastic cell line Fl 2. Using antibiotics we isolated stably expressed transformants. Among them, we selected one transformant, which barely expressed LacZ at steady state level but is induced to express upon treatment with BMP. Since it is known that BMP induced chondrogenic differentiation in vitro, BMP responsive promoter may reflect at least one aspect of chondrogenic differentiation. We named such transformants as pR5-1 cells. We next analyzed Lac~ Z expression by FACS. BMP treated pR5-l cells showed higher amount of LacZ positive cells than untreated cells, showing that FACS can be useful to collect cells in which the promoter is activated. We then prepared chondrocyte specific cDNAs. We used teratocarcinoma derived mesodermal cell line Cl as a source of RNA. We prepared cDNA libraries based on retrovirus vector plasmid and transfected them into a packaging cell line. Then we collected supernatant of those cells. We infected retrovirus cDNA library into pR5-1 cells. We, then, collected highly LacZ positive pR5-1 cells. We are now under way to isolate and characterize integrated genes.