| Project/Area Number |
11557038
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| Research Category |
Grant-in-Aid for Scientific Research (B).
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
内科学一般
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
OZAKI Shoichi Kyoto Univ Grad Sch Mde, Assoc. Prof., 医学研究科, 助教授 (00231233)
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| Co-Investigator(Kenkyū-buntansha) |
OHMOTO Yasukazu Institute of Otsuka Pharm., Chief Invest., 細胞工学研究所, 主任研究員
SUGINO Hiromu Tokushima University, Professor, 分子酵素学研究センター, 教授 (50211305)
OGAWA Yoshihiro Kyoto Univ Grad Sch Mde, Assist. Prof., 医学研究科, 助手 (70291424)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥13,300,000 (Direct Cost: ¥13,300,000)
Fiscal Year 2000: ¥4,800,000 (Direct Cost: ¥4,800,000)
Fiscal Year 1999: ¥8,500,000 (Direct Cost: ¥8,500,000)
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| Keywords | FRP / transgenic mice / transgene / type II collagen / PCR / FLAG epitope / DBA / 1 mouse / II型コラーゲンプロモーター / FLAGエピトープタグ / 1マウス / Iマウス |
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| Research Abstract |
To produce the transgenic mice, which can overexpress follistatin-related protein (FRP) specifically in the articular cartilage, the transgenes named Col2a1-mFRP and Col2a1-FLAG-mFRP were constructed. In both transgenes, mouse FRP (mFRP) cDNA was arranged downstream to the promoter of type II collagen. In Col2a1-FLAG-mFRP, the 5' nucleotide sequence of mFRP cDNA was modified to generate the protein bearing a FLAG epitope tag in the N terminus. To make sure that the transgenes could be transcribed under the regulation of type II collagen promoters, Col2a1-FLAG-mFRP was transferred to a mouse teratoma cell line ATDC5 in the developing stage to chondrocytes, and the transcribed mRNA was estimated. FLAG-mFRP mRNA was certainly detected by RT-PCR with its gene-specific primers. In this experiment producing transgenic mice, Col2a1-FLAG-mFRP of the two transgenes was adopted for the easy detection of the gene products by FLAG epitope tags. Col2a1-FLAG-mFRP was injected into embryos of DBA/1 (Crj) mice, and morphologically intact embryos were transferred to the uterine tubes of ICR mice. 310 embryos out of 344 transgene-injected ones were transplanted to eleven mice, and two mice became pregnant and bore four F0 offspring. RT-PCR analysis of genomic DNA with FLAG-mFRP-specific primers detected the transgenes in only one F0 mouse. This transgene-positive F0 mouse had almost the same phenotype as the wild type mouse. We are now producing F1 mice by mating this F0 mouse with wild DBA/1 (Crj) mice, and analyzing the transgenes in the born 53 F1 mice. As a control study, we performed arthritis-induction experiments in wild type mice with monoclonal antibodies to collagen (Arthritogenic mAb Cocktail, IBL), and succeeded in causing arthritis in all the tested mice. From now on, we will continue the transgene injection to the embryos in order to get more candidate strains of transgenic mice in this study. This project is now under way.
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