Development and establishment of practical method for production of transgenic goats

在美国和欧洲国家，用相对较小和易于处理的无趾龙来生产转基因家畜已经成为商业上的可行性。另一方面，日本还没有制定出可靠的生产转基因动物的方法。本项目的目的是开发实用的山羊TG技术。建立了山羊核移植供体培养细胞系(CPF-1)，分析了CPF-1的染色体数目和端粒长度与世代数的关系，评价了精子的冷冻保存，建立了山羊的体外受精/体外受精程序。对编码蛋白质因子的基因和/或cDNA进行了分子克隆的尝试，这些基因和/或cDNA可能用于山羊的基因工程，作为偶蹄类农场动物的模型，并取得了值得注意的成功结果。肌肉抑制素基因编码调控骨骼…发育的基因更多的肌肉和HMGI-C基因已经被成功克隆和序列分析，HMGI-C负责控制体型。还克隆了性别决定基因SRY和DAX1，并测定了它们的核苷酸序列。构建了反义小鼠Sry基因的表达载体，建立了转基因小鼠。采用CAGGS构建载体作为反义Sry反义反义基因的启动子/增强子复合体。共获得12只基因频率与性别表型一致的转基因小鼠。在来自创始人的F1中，对基因的初步PCR分析产生了结果，这让我们怀疑可能存在表型雌性的存在。进一步的验证性实验目前正在进行中。针对抗mSry核酶的开发，我们设计了几种核酶。检测了这些核酶对体外表达的Sry mRNA的作用，证明锤头状核酶和一种核酶-tRNA复合体能够有效地切割Sry mRNA。较少

Production of transgenic domestic animals in aitiodactyls with relatively small and facility of handling has become commercial feasibility in US and European countries. On the other hand, reliable methodology for the production of Tg animals has not yet been sufficiently worked out in Japan. The aim of present project is to develop practical Tg technologies in goats. The following researches had been carried out ; establishment of cultured cell line (CPF 1) as a donor for nuclear transplantation, analysis of chromosome numbers and relationship between telomere length and generation numbers in CPF 1, assessment of cryopreservation for spermatozoa and establishement of IVM/IVF procedures in goats. Attempts for molecular cloning of genes and/or cDNAs coding for protein factors potentially useful in genetic engineering of goats as a model of artiodactyl farm animals has been made and yielded noteworthy successful outcomes. The cDNAs coding for myostatin which regulates development of skele … More tal muscles, and HMGI-C responsible for the control of body-size have successfully been cloned and sequence-analyzed. Two sex determining genes, SRY and DAX1 have also been cloned and their nucleotide sequences were determined. Expression vectors for antisense murine Sry gene were constructed and Tg mice were produced. As the promoter/enhancer complex which drives the antisense -Sry trnasgene, CAGGS construct was employed. Twelve Tg mice were obtained as the founders where the genotypes coincided with the sexual phenotypes. In F1 derived from the founders, preliminary PCR analysis of the genes yielded results which led us to suspect the possible presence, of phenotypic females. Further confirmatory experiments are currently in progress. With regard to the development of anti-mSry ribozymes, we designed several ribozyme. The effects of these ribozymes against the Sry mRNA expressed in vitro were examined and evidence was obtained that the hammerhead ribozyme and one riobzyme-tRNA complex were capable of effectively cleaving the Sry mRNA. Less