| Co-Investigator(Kenkyū-buntansha) |
BALLING Rudolf DIRECTOR,GSF-Forchungszentrum Institut fuer Saeugetiergenetik (INSTITUTE OF MAMM, 所長
IMAI Kenji RESEARCH FELLOW,GSF-Forchungszentrum Institut fuer Saeugetiergenetik (INSTITUTE, 研究員
ARTZT Karen PROFESSOR,DEPT.OF ZOOLOGY,UNIVERSITY OF TEXAS AT AUSTIN,USA, 動物学, 教授
KANAME Tadashi ASSISTANT PROFESSOR,KUMAMOTO UNIVERSITY,SCHOOL OF MEDICINE, 医学部, 助手 (40264288)
阿部 訓也 熊本大学, 医学部, 助教授 (40240915)
KUNIYA Abe ASSOCIATE PROFESSOR,KUMAMOTO UNIVERSITY,SCHOOL OF MEDICINE
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| Research Abstract |
In this project, we try to establish an experimental approach coupling positional cloning and transgenic technologies for identification and isolation of mutated genes. Recent advances in genome analysis technologies enable us to isolate large size mammalian genomic DNA into YAC (Yeast artificial chromosome) or BAC (bacterial artificial chromosome) vectors. In combination with efficient genetic mapping techniques, it is feasible to map any mutation on mouse chromosome, and narrow down the region containing mutated gene to submega base range. However, identifying the relevant mutated gene can still be a formidable task, and a conventional method of identifying a gene by searching DNA sequence differences between mutant and wild-type may not always be possible. In those cases, alternative approach utilizing transgenic technologies would have potential to define the location of a gene on a large genomic DNA,i.e.introducing YAC or BACs containing genomic fragments harboring candidate genes
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into mutant mice and scoring for the rescue of the phenotype. We are now trying to identify mutated genes for developmental mutations such as t^<w5>, quaking both in the T/t complex of mouse Chromosome 17, and tk (tail kinks) on Chromosome 9. In each case, we have narrowed down the region where mutated gene resides to several hundred kb to 1 Mb. In order to find the mutated genes for these mutations, we first established the methodologies for production of transgenic mice carrying YAC or BAC DNA.YAC or BAC DNAs were prepared as follows ; an agarose plug containing YAC or BAC DNA was run on a pulsed field gel, and a band corresponding YAC/BAC clone waw excised and treated with agarase. After the enzyme digestion, released DNAs were washed and concentrated by a filtration apparatus. DNA solution of 5ng/mul was microinjected into mouse fertilized eggs. When a YAC clone with 650kb insert derived from the t^<w5> region was injected, we could establish three lines of transgenics carrying -270kb, -380kb and -450kb, respectively. Also, we have produced two transgenic mice, and found that both of mice contained intact, 150kb BAC clone derived from tail kinks region. Using these transgenic mice, we have begun breeding experiments to ask if these transgenes can rescue mutant phenotypes or not. Less
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