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

本项目旨在改进核移植生产克隆牛的技术，并将其推广应用。该项目的目标是用85个相同的胚胎生产26头克隆牛，其中移植给受体牛的妊娠率估计为30%。通过该项目，所有研究人员都达到了目的，核移植胚胎克隆技术得到了显著提高。结果表明：1)连续核移植系统获得了约30枚(最多43枚)完全相同的胚胎，移植到受体牛体内后获得了存活的胚胎；2)冷冻保存的不明原因移植胚胎可以产生存活的胚胎，冷冻后的未受精卵可以作为受体细胞质；3)在共培养系统中，小鼠胚胎成纤维细胞对核移植胚胎的发育具有很强的支持能力；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，6)本课题实现了通过核移植建立有效克隆技术的目的，通过核移植共获得了40只活体幼虫。

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.