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Constuction of a System for the Deployment of Advanced Biotechnology to Riminants of Industrial Importance.

构建一个将先进生物技术部署到具有工业重要性的问题的系统。

基本信息

批准号：
04556040
负责人：
TACHI Chikashi
金额：
$ 12.22万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1994
项目状态：
已结题

项目摘要

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 … More 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

在研究期间，取得了以下主要成果：1)以lambda噬菌体为载体，成功构建了柴山羊（Capra hircus var Shiba）基因组DNA文库。通过筛选文库，克隆了具有启动子区和3^'非翻译区的山羊SRY基因，并对其进行了测序。2)已知哺乳动物的c-kit基因编码酪氨酸激酶受体，酪氨酸激酶受体在维持原始生殖细胞、造血细胞和色素细胞的存活中起重要作用。我们克隆了柴山羊的c-kit cDNA，包括ORF的完整长度和3^' UTR区。3)研究了不同时间间隔联合注射GnRH、PGF2alpha、FSH和HCG诱导反刍动物超排卵的条件。结果表明，第0天给予GnRH，第2天给予PGF2alpha，第4、5、6天给予FSH，第7天给予HCG可有效诱导超排卵。然而，超额覆盖的总数比最初预期的要少得多。这方面的进一步研究是有必要的。4)作为产生转基因牛的基础研究的一部分，我们研究了奶牛（Bos taurus）卵母细胞体外成熟、体外受精和早期胚胎体外培养的各种条件。对于体外受精，用于精子稀释的培养基的组成和公牛个体精子质量的变化是影响体外受精结果最显著的因素。5)我们试图开发一种有效的程序来诊断外源引入的DNA结构在转移到受体动物子宫之前成功地整合到基因组中的转基因胚胎。我们描述了一种在原核注射外源DNA后高效选择转基因胚胎的改进方法。该方法基于从胚胎中提取的DNA与限制性内切酶和PCR相结合的处理。我们的程序包括在PCR前用玻璃乳回收消化的DNA。当外源导入小鼠胚胎的DNA序列未整合到基因组中时，将其与Dpn I和Bal 31联合处理消化，随后的PCR分析仅在1.5%（1/65）的检测病例中产生了注射DNA序列的DNA片段。少