| Project/Area Number |
08557095
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Morphological basic dentistry
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| Research Institution | Osaka University |
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Principal Investigator |
KURISU Kojiro Osaka University Faculty of Dentistry, Professor, 歯学部, 教授 (50028346)
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| Co-Investigator(Kenkyū-buntansha) |
SATO Katsuhiko Chugai Pharmaceutical Co., LTD., 研究者
TABATA Makoto Osaka University, Faculty of Dentistry, Assistant, 歯学部, 助手 (20243248)
KATO Joji Osaka University, Faculty of Dentistry, Assistant, 歯学部, 助手 (90243245)
IWAMOTO Masahiro Osaka University, Faculty of Dentistry, Instructor, 歯学部, 講師 (30223431)
WAKISAKA Satoshi Osaka University, Faculty of Dentistry, Associate Professor, 歯学部, 助教授 (40158598)
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| Project Period (FY) |
1996 – 1997
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| Keywords | Orofacial tissue / Gene / Gene multiplication / Rabbit / Morphogenesis / Development / Tissue differentiation / cDNA library |
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| Research Abstract |
Subtractive hybridization methods are used to detect genes that are expressed in a specific tissue or cell but not in the other. Usually cDNA called "tester" that contains differentially expressed genes is hybridized with an excess amount of RNA called "driver" that does not contain such genes. As a result, differentially expressed cDNA in enriched in unhybridized cDNA fraction. Although traditional subtractive hybridization methods have been successfully used in some cases, they require a lot of tester and driver mRNA.Therefore, it has been difficult to identify differentially expressed genes when only small amount of tissue (e.g.mouse tooth bud etc.) is available as a starting material.
In the present study, we constructed chondrocytes specific subtraction cDNA library by two different methods : [1] conventional single strand subtraction (tester cDNA-driver mRNA) and [2] double strand cDNA subtraction (tester dscDNA-driver dscDNA). In both methods, we used PCR for amplifying tester cDNA.We found the quality of those libraries was similar but the amount of tester mRNA that was required for library construction was much smaller in the method [2] than that of [1]. In both libraries, most of cDNA clones encodes 3' untranslated sequence plus partial sequence and the average size of inserted cDNAs was about 550bp. We speculated this was due to the limitation of PCR amplification. Although partial cDNA may be useful as a probe to examine expression pattern of the gene in the tissue, It is necessary screen the unsubtracted library to obtain full length cDNA.
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