1992 Fiscal Year Final Research Report Summary
Molecular cloning and basic study of bone inducing factors for their clinical use.
| Project/Area Number |
03670937
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
外科・放射線系歯学
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| Research Institution | TOKYO MEDICAL AND DENTAL UNIVERSITY |
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Principal Investigator |
IWAKI Hiroshi Tokyo Med.& Dent.Univ.,1st Dept.of Oral & Maxillofacial Surg., Faculty of Dentistry, Research Associate, 歯学部, 助手 (70107308)
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| Co-Investigator(Kenkyū-buntansha) |
MOMOSE Fumio Tokyo Med.& Dent.Univ., 1st Dept.of Oral & Maxillofacial Surg., Faculty of Denti, 歯学部, 助手 (60157849)
OIDA Shinichiro Tokyo Med.& Dent.Univ., Biochemistry, Faculty of Dentistry Research Associate, 歯学部, 助手 (10114745)
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| Project Period (FY) |
1991 – 1992
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| Keywords | Bone Morphogenetic Protein(BMP) / Polymerase Chain Reaction(PCR) / Recombinant Protein |
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| Research Abstract |
To understand the mechanism of biological actions of bone inducing factors, we attempted to clone the genes for these factors.
According to the human BMP(Bone Morphogenetic Protein)-2/4 and mouse BMP-6(Vgr-1) nucleotide sequences, oligonucleotide primers were prepared. Messenger RNAs were isolated from neonatal mouse tissue and human osteosarcoma cell line MG63, and used for the synthesis of complementary DNAs (cDNAs) with reverse transcriptase. By PCR(Polymerase Chain Reaction) using these primers and cDNA templates, we have cloned cDNAs for mouse BMP-2, mouse BMP-6, and human BMP-4.
To isolate full length of BMP cDNAs, various cDNA libraries were screened with the cDNA probes cloned by PCR. A positive clone detected in the screening of a human placenta cDNA library was subcloned into a plasmid vector and subjected to nucleotide sequencing. The result was that the positive clone was found to be a full length cDNA for human BMP-4. Using the same method, an almost full length human BMP-6 cDNA was also isolated from a human lung small cell carcinoma(NCL-H69) cDNA library.
Furthermore, we attempted to synthesize the human BMP-4 recombinant protein. The human BMP-4 cDNA was transferred into two derivatives of the E.coli expression vector, and a beta - Galactosidase-human BMP-4 fusion protein and a Glutathione-S- transferase-human BMP-4 fusion protein were obtained. Osteoinductive activities of these fusion proteins are now under investigation.
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