1987 Fiscal Year Final Research Report Summary
Introduction of foreigh gene into fertilizes mammalian egg by microinjection
| Project/Area Number |
61480075
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
畜産学(含草地学)
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| Research Institution | Ibaraki University (1987-1988)
The University of Tokyo (1986) |
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Principal Investigator |
SHODA Yoichi Ibaraki University Department of AgricultureProfesson, 農学部, 教授 (80011815)
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| Co-Investigator(Kenkyū-buntansha) |
IKE fumio The Institute of Physical and Chemical Research Research Atudcut, 動物薬理, 研究員 (40183157)
SAKAI Swnkiti University of Tokyo Faculty of Agriculture Assisrant Professon (KAWAZOE,Yosh), 農学部, 助教授 (80114487)
KOHMOTO Kaoru University of Tokyo Faculty of Agriculture Proflesson, 農学部, 教授 (30011894)
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| Project Period (FY) |
1986 – 1987
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| Keywords | Fertilized egg / Microinjction / Gene transfer / Effect of EDTA / Effect of DNA concentration / Human growth hormone / メタロチオネーンI |
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| Research Abstract |
We conucted the introduction of foreign genes intto fertilizes mammalian eggs by microimjection. The plasmid cosists of the 5-flanking region of the metallothionein-I gene, the structural gene of human growth hormono and a vector sequence of pBR-322. The genes used for microinjection were 2.5-kb DNA fragments isolated after digestion with Bst EII and Eco RI. For genetical analysis, the 1.1-kb Pvu II fragment of the structural gene of human growth hormone was used as a probe. Fertilized eggs were obtained from the ampullary regions of 4-week-old C57BL/6N mice, which were seperovulated with PMSG and hCG and mated with mature male mice. DNA solution (1-2 p1) was microinjected to these eggs. Injected eggs were cultured overnight in the CO_2 incubator and cleaved embryos were transfered into the pseudopregnat Jc:ICR mice. Integration of the injected genes was examined by the dot-blot and the Southern blot hybtidization of DNA obtained from fetuses or vaible offspring. (1) Effect of EDTA con
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centration in the solution on development of injected embryos. From 72 to 89 percents of embryos which had received low concentration of EDTA were cleaved and were a little fewer than intact control embryos. Percentage of embryos which developed to the blastocyst stage decreased as the concentration of EDTA increased. At 5 mM of EDTA., extremely few embryos developed to the blastocyst stage (3.7%). (2) Effect of DNA concentration on development of imjected embryos. At higher concentrations. the fewer embryos cleaved. The implantation ratios of embryos which had received 100 to 1000 copies/p1 were nearly the same as to the intact control. Extremely few embryos, received 10000 copies/p1, were implanted and survived after implantation (less than 5 %). (3) Production of transgenic mice. By microinjection of 1000 copies/p1 of the gene to male pronuclei, 109 cleaved embryos were obtained, transferred into oviducts foster mothers, and 19 offspring were born. Seven offspring were dead before weaning. Only 12 weaned mice were analyzed for the quantity and the size of inegration of the injected DNA to somatic cells. The transferred gene was detected in one mouse by 150 to 200 copies per cell with the same length to the introduced. Less
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[Publications] Kawamura, K., Enami, J., Enami, S., Koezuka, M., Kohmoto, K., Koga, M.: "Growth and morphogenesis of mouse mammary epithelial cells cultures in vitro." Proceeding of Japanese Academy. 62b. 5-8 (1986)
Description
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