Preparation of knockout Mouse of a transregulatory fector, OCZF which is specifically expressed inosteoclasts
| Project/Area Number |
15591970
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional basic dentistry
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| Research Institution | Saga University (2004)
佐賀医科大学 (2003) |
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Principal Investigator |
KUKITA Akiko Saga University, Medicine, Associate Professor, 医学部, 助教授 (30153266)
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| Co-Investigator(Kenkyū-buntansha) |
KUKITA Toshio Kyushu University, Dentistry, Associate Professor, 大学院・歯学研究院, 助教授 (70150464)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2004: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2003: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | osteoclast / transcriptional factor / knockout / ES cell |
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| Research Abstract |
To investigate the role of transcriptional regulator, OCZF(Osteoclast zinc finger protein) specifically expressed in osteoclasts, we isolated a genome clone of mouse homologue of OCZF, LRF(Leukocyte-related factor) from mouse BAG clone ES cell genome library and prepared targeted vector which had neomycin resistant gene. ES cells were introduced by targeted vector by electroporation and G418 was added. After selection for 9 days, 500 resistant ES clones were picked up. ES resistant cells were analyzed by PCR and southern analysis and 6 LRF (-/+) ES cell clones were obtained. We then increased the concentration of G418 and obtained LRF(-/-) ES cells to analyze the possibility of osteoclast differentiation in the absence of LRF gene.
During the course of the experiments, we are informed that LRF knockout mouse are already prepared by American group and mouse is embryonic lethal. We then determined to collaborate with American group and Japanese group to use LRF knockout mouse.
We also performed to knockout OCZF/LRF gene by using siRNA in vitro. We analyzed the condition which is easily introduced RNA and DNA into mouse macrophage cell line, RAW264 and found that RAW 264 cells were introduced by RNA up to 80 % of the cell population by using Nucleofector. Several small RNAis were prepared to repress the expression LRF gene in RAW 264 cells. Introduction of RNAi into RAW 264 cells stimulated with RANKL markedly inhibited osteoclast differentiation of RAW 264 cells.
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Report
(3 results)
Research Products
(26 results)
