| Project/Area Number |
10356010
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| Research Category |
Grant-in-Aid for Scientific Research (A).
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Applied animal science
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| Research Institution | THE UNIVERSITY OF TOKYO |
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Principal Investigator |
TOJO Hidcaki The University of Tokyo, Graduate School of Agricultural and Life Sciences, Professor, 大学院・農学生命科学研究科, 教授 (20041668)
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| Co-Investigator(Kenkyū-buntansha) |
YAMANOUCHI Keitarou The University of Tokyo, Graduate School of Agricultural and Life Sciences, assistant professor, 大学院・農学生命科学研究科, 助手 (70272440)
NAITO Kunihiko Yamanouchi The University of Tokyo, Graduate School of Agricultural and Life Sciences, Associate Professor, 大学院・農学生命科学研究科, 助教授 (20188858)
小野寺 節 東京大学, 大学院・農学生命科学研究科, 教授 (90012781)
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| Project Period (FY) |
1998 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥30,600,000 (Direct Cost: ¥30,600,000)
Fiscal Year 2000: ¥7,500,000 (Direct Cost: ¥7,500,000)
Fiscal Year 1999: ¥10,100,000 (Direct Cost: ¥10,100,000)
Fiscal Year 1998: ¥13,000,000 (Direct Cost: ¥13,000,000)
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| Keywords | co-injection / DNA microinjection / EGFP / cre / loxP / the Dpn I-Bal 31 / restriction enzyme / Transgeic animals / トランスジェニック / マウス胚 / マーカー遺伝子 |
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| Research Abstract |
1. We investigated whether or not co-injection of foreign DNA constructs with restriction endonuclease into the pronucleus of mouse zygotes would improve the integration frequencies of foreign DNA into the host genome. Two kinds of DNA constructs that have no Eco RI site in their sequences were used for co-microinjection. Successful transgenesis of co-injected embryos was identified by the Dpn I-Bal 31 digestion method for single embryos and by PCR method for pups born, respectively. The overall efficiency for the integration of foreign DNA in single embryos and live-born pups obtained by the co-injection procedures were 17.9 % compared with 9.1% obtained by the injection of DNA alone. The results suggest that co-injection of foreign genes with restriction enzyme may elevate the integration rate of foreign genes into host genomes. 2. We developed a method for obtaining the high-efficient production of transgenic mouse by selecting EGFP-expressing embryos prior to embryos transfer.
The C
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AG/EDPP-WAP/hGH fusion gene was microinjected into the pronucleus of zygotes, and then after in vitro culture, morula or blastocyst-satge embryos were observed under a fluorescent microscope for fluorescence. Approximately 86% transgenesis were derived from the transfer of the embryos with uniform EGFP-expression. The results showed that this method should contribute to the high efficient production of transgenic animlas 3. Cre expressing plasmid and a transgene (CMV/LacZ gene) flanked by two pseudo-loxM5 sites were co-microinjected into the pronucleus of mouse fertilised oocytes. The injected eggs were transferred into the pseudo-pregnant mice. Recombination products were investigated by PCR.The transgenesis efficiency was up to 25% (4/16). The site-specific integration of the transgene into the endogenous pseudo-loxM5 sites was found in all of the transgenic pups by PCR analysis using specific primers. These results demonstrate that Cre/pseudo-loxM5 integrative recombination system is an efficient and simple strategy to target an endogenous lox-like site in the early mammalian embryos. Less
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