Exploitation of new methods for efficent production of transgenic animals

转基因技术为研究基因在细胞和生理功能中的表达调控提供了强有力的工具。转基因农场动物的产生是为了改善家畜的产品或生产力以及对动物疾病的抗性。(1)在家畜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，由于外源DNA在宿主DNA中的整合率较低，转基因家畜的整体效率远低于小鼠，本研究旨在提高转基因家畜的生产效率。(2)为了提高外源DNA在宿主DNA中的整合率，将带有DNA的限制性内切酶显微注射到合子原核中，诱导宿主基因组DNA的断裂，增加外源DNA整合到宿主DNA中的机会。在10 ~ 10 IU的8种浓度<-4>的EcoRI中，10 ~ 10 IU<-1.5>和10 IU对宿主基因组DNA的适当断裂效果最好。建立了转基因胚的筛选方法<-2>，用Dpn I和Bal 31酶消化受体胚胎中未整合到宿主基因组中的DNA片段。结果表明，该方法可用于转基因胚的筛选。(3)作为提高转基因家畜生产率方法学的基础性试验，本研究对从牛卵巢卵泡中克隆的牛卵母细胞进行了体外成熟、受精和培养的研究。结果表明，冻精个体差异对体外受精成功率有显著影响。

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.