| Project/Area Number |
01044114
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Institution | Osaka University (1990-1991)
Kumamoto University (1989) |
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Principal Investigator |
SHINADA Kazunori Research Institute for Microbial Diseases, 微生物病研究所, 教授 (40037354)
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| Co-Investigator(Kenkyū-buntansha) |
ROBERTSON El コロンビア大学, 医学部, 助教授
GOTTESMAN Max E. Institute of Cancer Research, Columbia University, 癌研究所, 所長
MAEDA Shuichiro Kumamoto Medical School, 医学部, 助教授 (10117244)
NISHIGUCHI Seiji Research Institute for Microbial Diseases, 微生物病研究所, 助手 (90237686)
ROBERTSON Elizabeth J. College of Physicians and Surgeons of Columbia University
瀬戸山 千秋 熊本大学, 医学部, 講師 (60040250)
瀧原 義宏 大阪大学, 微生物病研究所, 助手 (60226967)
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| Project Period (FY) |
1989 – 1991
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| Project Status |
Completed (Fiscal Year 1991)
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| Budget Amount *help |
¥10,600,000 (Direct Cost: ¥10,600,000)
Fiscal Year 1991: ¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 1990: ¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 1989: ¥4,000,000 (Direct Cost: ¥4,000,000)
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| Keywords | Gene targeting / Homologous recombination / Embryonic stem cell / Mouse model for human diseases / Chimeric mouse / Prealbumin / Amyloidosis / Hereditary diseases |
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| Research Abstract |
1. We constructed a sequence replacement vector to introduce an inactivating neo^r marker into the mouse genomic prealbumin (PA) gene by homologous recombination. The targeting vector consisted of a 5.9 kb DNA fragment derived from the mouse PA genomic locus and the 1.1 kb neo^r gene from pMCINeo. In this vector, the mouse PA DNA fragment was separated by the neo^r gene into two segments, one was small (1.3 kb) and the other was large (4.6 kb). A counterselectable HSV-tk gene was placed at the end of the long region of homology.
2. This vector was electroporated into cultured mouse embryonic stem (ES) cells, which were subsequently selected with G418 and gancyclovir. Among the 132 colonies obtained, 6 homologous recombinants were identified by PCR and Southern blotting. The ratio of homologous recombination to random integration was approximately 1 : 80.
3. These ES cells were used to generate chimeric mice by injection into blastocysts. One of these chimeras has shown germline transmiss
… More
ion of the disrupted PA gene. Mice heterozygous for the PA gene appeared to be phenotypically normal. Heterozygous mice were crossed to try to generate homozygotes. Homozygous PA deficient mice were found at the expected Mendelian frequencies and appeared to be phenotypically normal. We are now in the process of establishing transgenic mouse lines carrying the human mutant PA gene which is responsible for familial amyloidotic polyneuropathy (FAP) in the homozygous mutant background.
4. We constructed a sequence replacement vector to introduce a point mutation which is responsible for FAP into the mouse PA gene by homologous recombination. The vector consisted of the following fragments in the order of their sequences : 5'-flanking region of the mouse PA gene - the first exon - the first intron - the second exon which has the point mutation - a part of the second intron* MC-neo/MC-tk gene - the second intron which contains the * fragment the third exon - a part of the third intron. This vector was electroporated into the ES cells and a total of 768 G418^r colonies in 128 pools were analyzed by allele-specific PCR, but no homologous recombinants were detected. Less
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