2000 Fiscal Year Final Research Report Summary
IDENTIFICATION OF DISEASE GENE USING BAC TRANSGENIC MICE
| Project/Area Number |
11694296
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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 | 一般 |
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| Research Field |
Laboratory animal science
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| Research Institution | KUMAMOTO UNIVERSITY |
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Principal Investigator |
YAMAMURA Kenichi KUMAMOTO UNIVERSITY, INSITUTE OF MOLECULAR EMBRYOLOGY AND GENETICS, PROFESSOR, 発生医学研究センター, 教授 (90115197)
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| Co-Investigator(Kenkyū-buntansha) |
OHBO Kazuyuki KUMAMOTO UNIVERSITY, INSTITUTE OF MOLECULARA EMBRYOLOGY AND GENGETICS, ASSITATNT PROFESSOR, 発生医学研究センター, 助手 (70250751)
ARAKI Kimi KUMAMOTO UNIVERSITY, INSTITUTE OF MOLECULARA EMBRYOLOGY AND GENGETICS, ASSISTANT PROFESSOR, 発生医学研究センター, 助手 (90211705)
ABE Kuniya KUMAMOTO UNIVERSITY, INSTITUTE OF MOLECULARA EMBRYOLOGY AND GENGETICS, ASSOCIATE PROFESSOR, 発生医学研究センター, 助教授 (40240915)
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| Project Period (FY) |
1999 – 2000
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| Keywords | BAC / YAC / TRANSGENIC MOUSE / OUAKING / T COMPLEX / MICROINJECTION |
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| Research Abstract |
Chromosome should be replicated without any loss of DNA at the end of chromosome and each one chromosome should be separated into daughter cells while cell division. To accomplish this process, at least three elements, replication origin, telomere, and centromere, are required. In yeast and E.coli, these elements are well characterized and are already cloned. Using these elements, it is now possible to construct artificial chromosome such as yeast artificial chromosome (YAC) or bacterial artificial chromosome (BAC). These artificial chromosomes can be used for isolating a large fragment of DNA spanning to a few hundreds of kb in BAC and a few magabase in YAC.As YAC is quite unstable, use of BAC is becoming more popular. We have tried to establish a method to produce transgenic mice by microinjecting BAC into fertilized eggs. We found that isolation of BAC can be done by pulse field gel electrophoresis, removal of agarose gel fraction containing BAC with about 100 to 200 kb DNA, and digestion of agarose with agarase. The optimum concentration of BAC DNA for microinjection was 1 to 1.5 ng/μl. Although the number of live born mice is lower than that in injection with normal DNA, the overall efficiency of transgenic production was the same and 10 to 60 % of live mice were transgenic. As BAC9 of about 165kb size seems to contain whole qkI gene, we injected these BAC9 and established two lines of transgenic mice. These mice were mated with quaking mice to produce transgenic mice with quaking background. These transgenic mice did not develop quaking phenotype. BAC containing T gene could also rescue the T phenotype. These results clearly suggest that BAC transgenesis can be a powerful tool to test whether the BAC used for transgenesis contains a candidate gene.
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