| Project/Area Number |
04556040
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| Research Category |
Grant-in-Aid for Developmental 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 | The University of Tokyo |
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Principal Investigator |
TACHI Chikashi The University of Tokyo, School of Agriculture and Life Sciences, Dept. Animal Sciences, Professor, 農学部, 教授 (30011711)
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| Co-Investigator(Kenkyū-buntansha) |
TOJO Hideaki The University of Tokyo, School of Agriculture and Life Sciences, Dept. Animal S, 農学部, 助教授 (20041668)
MORI Yuji The University of Tokyo, School of Agriculture and Life Sciences, Associate Prof, 農学部, 助教授 (40157871)
SAWASAKI Tohru The University of Tokyo, School of Agriculture and Life Sciences, Professor, 農学部, 教授 (00012047)
KOHMOTO Kaoru The University of Tokyo, School of Agriculture and Life Sciences, Professor, 農学部, 教授 (30011894)
TAKAHASHI Michio The University of Tokyo, School of Agriculture and Life Sciences, Professor, 農学部, 教授 (30011943)
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| Project Period (FY) |
1992 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥19,100,000 (Direct Cost: ¥19,100,000)
Fiscal Year 1994: ¥5,000,000 (Direct Cost: ¥5,000,000)
Fiscal Year 1993: ¥6,700,000 (Direct Cost: ¥6,700,000)
Fiscal Year 1992: ¥7,400,000 (Direct Cost: ¥7,400,000)
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| Keywords | Genomic Library of Shiba goats / Biotechnology of Farm Animals / SRY / c-kit / Super-ovulation / in vitro Ovum Maturation / in vitro Fertilization / Methylation / DpnI / Bal3I / バイオ技術 / 反芻類家畜 / シバヤギ / 反屈類家 / 胚性幹細胞 / トランスジェニック |
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| Research Abstract |
During the period of research, following mjor results were obtained. 1)A genomic DNA library of Shiba goat (Capra hircus var Shiba) was successfully constructed using lambda phage as the vector. Caprine SRY gene coplete with the promoter region and 3^' untranaslated region was cloned and sequences by screening the library. 2)The c-kit gene in mammals has been known to code for a tyrosine-kinase receptor which plays crusial roles in the maintenance of survival of primordial germ cells, hematopoietic cells and pigment cells. we cloned the c-kit cDNA of Shiba goat including the complete length of ORF and the 3^' UTR region. 3)Conditions for the induction of superovulation in ruminants were investigated by injection a combination of GnRH,PGF2alpha, FSH and HCG at different time intervals. Results were obtained suggesting that a regimen of administration of GnRH on Day 0, PGF2alpha on Day 2, FSH on Day 4,5,6 and HCG on Day 7 was effective in inducing the superovulation. However, the total n
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umber of ovarecovered was much smaller than that originally expected. Further studies in this regard are in order. 4)As a part of basic research leading to the generation of transgenic cattle, we examined various conditions for in vitro maturation of oocytes, in vitro fertilization, in vitro culture of early embryos in cow (Bos taurus). As for the in vitro fertilization, the composition of the medium used for the dilution of sperm and the variations in the quality of sperm of individual bulls werre found to affect the outcome of IVF most significantly. 5)We attempted to develop a procedure effective in diagnosing the transgenic embryos where the exogenously introduced DNA constructs were successfully incorporated into the genome prior to the transfer to the uteri of recipient animasl. We described an improved method for efficient selection of transgenic preimplantation-embryos after pronuclear injection of exogenous DNA.The method is based on the combined treatment of DNA extracted from the embryos with restriction enzymes and PCR.Our procedure included recovery of the digested DNA with glassmilk prior to PCR.When the DNA sequences exogenously introduced into the mouse embryos were not integrated into the genome they were digested by combined treatment with Dpn I and Bal 31, and subsequent PCR analysis generated DNA fragments of the injected DNA sequence in only 1.5% (1/65) of cases examined. Less
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