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Exploitation of new methods for efficent production of transgenic animals

转基因动物高效生产新方法的开发

基本信息

批准号：
05660323
负责人：
TOJO Hideaki
金额：
$ 1.41万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05660323/
关键词：
Transgenic Bovine embryos PCR Farm animals 制限酵素

项目摘要

Transgenic technology has provided powerful tools for the examination of the regulation of gene expression in cellular and physiological funtions. Transgenic farm animals havd been generated for improved products or productivity of domestic animals and resistance to animal diseases. (1) The most common method used for gene teansfere in farm animals is microinjection of DNA into pronuclei of zygotes. However, overall efficiency of transgenic farm animals were extreamely lower than that of miceowing to low integration of foreing DNA into host DNA.The present study was designed to improve the efficiency of generation of transgenic farm animals. (2) To improve the integration rate of for eign DNA into host DNA,the restriction enzyme with DNA was microinjected into the pronuclei of zygotes to induce the breaks in host genome DNA which increase the opportunity of integration of foreign DNA inothost DNA.In 8 concentrations from 10^<-4> to 10IU of EcoRI,The 10^<-1.5> and 10^<-2> IU were most effective to appropriate break in host genome DNA.To establish the method for selecting the transgenic embryos before they are transffered to the recipient, DpnI and Bal 31 enzymes were used for digestion of DNA fragement that not integrated into the host genome in the embryos. After this treatment, integrated gene was amplified by PCR.The results showed that this method was useful for selection of transgenic embryos. (3) As a basic experiment for methodological improvement of production rate of transgenic farm animals, the in vitro maturation, fertilization and culture of bovien oocytes cololected from the ovarian follicles of carcus were sutdied. It was found that individula differences in frozen semen remarkably influenced on the successful efficiency of in vitro fertilization.

转基因技术为检查细胞和生理功能中基因表达的调节提供了有力的工具。转基因农场动物的产生是为了提高家畜的产品或生产力以及对动物疾病的抵抗力。 (1) 在农场动物中进行基因转移最常用的方法是将DNA显微注射到受精卵的原核中。然而，由于外来DNA与宿主DNA的整合程度较低，转基因家畜的总体效率远低于小鼠。本研究旨在提高转基因家畜的产生效率。 (2)为了提高外源DNA与宿主DNA的整合率,将带有DNA的限制性内切酶显微注射到受精卵原核中,诱导宿主基因组DNA断裂,增加外源DNA整合到宿主DNA中的机会。EcoRI在10^<-4>至10IU的8个浓度中,10^<-1.5>和10^<-2>IU最有效。宿主基因组DNA中适当的断裂。为了建立在移植给受体之前选择转基因胚胎的方法，使用DpnI和Bal 31酶消化胚胎中未整合到宿主基因组中的DNA片段。处理后，通过PCR扩增整合基因。结果表明，该方法可用于转基因胚胎的选择。 (3)作为提高转基因家畜生产效率的方法学基础实验，对从家畜卵泡中采集的牛卵母细胞的体外成熟、受精和培养进行了研究。研究发现冷冻精液的个体差异显着影响体外受精的成功率。