|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1995 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1994 : ¥1,000,000 (Direct Cost : ¥1,000,000)
To reveal the mechanism of action of Bone Morphogenetic Protein (BMP), we have examined the genes induced by BMP in the early stage of the response, which may control the action of BMP,using the differential display method in this study.
We used Osteogenic Protein-1 (OP-1, BMP-7) as BMP,because highly purified recombinant OP-1 was available from Creative BioMolecule Inc. Examination of the effect of OP-1 on several cell lines revealed that OP-1 enhances alkaline phosphatase activity, one of the maker of osteoblastic characteristics, in the culture of osteoblastic MC3T3-E1 cells from mouse calvaria and stromal ST-2 cells from mouse bone marrow. After the treatment of OP-1 to these cells for 24hours, total RNA was extracted with AGPC method and reverse transcription was performed. According to the instruction of the kit for differential display method, polymerase chain reaction (PCR) on the resulted cDNA was performed with 20 sets of primer combination under the presence of 35S-dATP.The amplified cDNAs were electophoretically separated on a 6% sequence gel. Analysis of bands on the auto radiogram revealed that 66 cDNA fragments from MC3T3-E1 and 11 cDNA fragments from ST-2 were expressed differentially with or without the treatment of OP-1. cDNAs from MC3T3-E1 were subcloned and sequences were analyzed. There were a few clones which have homology with ubiquitin, SP-1, etc.but most of them were not identified clearly because the acquired sequences were only 200 to 300 base in 3'end region. Northern analysis was performed using these cDNA clones as a probe, but expression of most of them did not have any difference between with and without the treatment of OP-1. Therefore, we assumed that many cDNA clones obtained by differential display methods were psudepositive. However, we got two cDNA clones which show apparent difference on Northern analysis from ST-2. Sequencing analysis is on procedure.