STUDIES ON DEVELOPMENT OF PRACTICAL TECHNIQUE FOR PRODUCING CLONE COWS BY NUCLEAR TRANSFER

本项目旨在改进核移植克隆牛生产技术，并将其应用于实际生产。该项目的目标是从85个相同的胚胎中培育出26头克隆牛，其中移植给受体奶牛后的受孕率估计为30%。在整个项目期内，所有研究者都达到了他们的目的，核移植胚胎克隆技术有了显著的提高。结果表明：1)通过连续核移植系统获得了30个（最多43个）相同的胚胎，并将这些胚胎移植给受体奶牛后获得了活的幼崽。2)结果表明，低温保存的未受精卵可作为受体细胞质。3)在共培养体系中，小鼠胚胎成纤维细胞对核移植胚胎的发育表现出较高的维持能力。4)建立了由重复电脉冲组成的有效激活系统，提高了核移植卵的发育能力。5) DNA聚合酶I抑制剂Aphidicolin用于同步供核细胞周期，可有效提高核移植卵的发育能力。本项目实现了建立一种有效的核移植克隆技术的目的，通过核移植共产生了40只活苗。

The present project was conducted to improve techniques for producing clone cattle by nuclear transfer and to apply for practical use it. The goal of the project was to produce 26 clone cattle from 85 identical embryos, in which the pregnancy rate after transfer to recipient cows was estimated as 30%. Through the project term, all investigators have achieved their aim and the technique for embryo cloning by nuclear transfer was significantly improved. The results are summarized as follows :1) We showed that about 30 (maximum 43) identical embryos were produced by the serial nuclear transfer system, and live young were obtained from such embryos after transfer to recipient cows.2) It was showed that live young can be produced from cryopreserved unclear transferred embryos, and that cryopreserved unfertilized eggs can be used as recipient cytoplasm.3) In co-culture system, mouse fetus fibroblast cells showed high sustaining ability for development of nuclear transferred embryos.4) Effective activation system that consisted of repetitive electric pulses was established, by which the developmental ability of nuclear transferred eggs was improved.5) Aphidicolin, DNA polymerase I inhibitor, which was used for synchronizing cell cycle of donor nuclei, was effective to improve developmental ability of nuclear transferred eggs.6) The present project has achieved the aim that is establishment of an effective cloning technique by nuclear transfer, and total of 40 live young were produced by nuclear transfer.