Development and establishment of practical method for production of transgenic goats

转基因山羊生产实用方法的开发和建立

• 批准号: 11356008
• 负责人: TACHI Chikashi
• 金额: $ 23.1万
• 依托单位: AZABU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11356008/
• 关键词: goart; transgenic; transgenic technologies; telomere length; myostatin; Sry; antisense; riobzyme; 精子の凍結保存; 体外受精; 卵成熟; DAX1; myostatin; HMGI-C; トランスジェニックマウス

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

在美国和欧洲国家，生产相对较小且易于操作的转基因缺趾家畜已具有商业可行性。另一方面，日本尚未充分制定生产Tg动物的可靠方法。本项目的目的是在山羊中开发实用的Tg技术。本研究对山羊体外培养细胞系CPF-1的建立、CPF-1的染色体数目、染色体端粒长度与代数的关系、精子冷冻保存效果的评价以及山羊体外成熟/体外受精（IVM/IVF）技术的建立进行了研究。已经尝试对编码蛋白质因子的基因和/或cDNA进行分子克隆，所述蛋白质因子在山羊作为偶蹄动物农场动物的模型的遗传工程中潜在有用，并且产生了值得注意的成功结果。肌肉生长抑制素的cDNA编码调控胚胎发育 ...更多信息 已经成功地克隆和序列分析了负责控制身体大小的HMGI-C。另外还克隆了两个性别决定基因SRY和DAX 1，并测定了它们的核苷酸序列。构建了反义Sry基因的表达载体，制备了Tg小鼠。作为驱动反义Sry转基因的启动子/增强子复合物，使用CAGGS构建体。获得12只Tg小鼠作为建立者，其中基因型与性表型一致。在来自创始人的F1中，基因的初步PCR分析产生的结果使我们怀疑可能存在的表型女性。目前正在进行进一步的验证性实验。在抗mSry核酶的开发方面，我们设计了几种核酶。研究了这些核酶对体外表达的Sry mRNA的影响，并获得了锤头状核酶和一种riobzyme-tRNA复合物能够有效切割Sry mRNA的证据。少

项目成果

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Kim CH：“ZP 分子在小鼠胚胎输卵管运输中的重要作用。”Mol Reprod Dev。

Seng S: "Increased susceptibility to Toxoplasma gondii infection in SAG-1 transgenic mice"Intern J Parasitol. 29. 1433-1436 (1999)

Seng S：“SAG-1 转基因小鼠对弓形虫感染的易感性增加”Intern J Parasitol。

Shimizu C: "Progression of T cell lineage restriction in the earliest subpopulation of murine adult thymus visualized by the expression of 1ck proximal promoter activity"Intern Immunol. 13. 105-117 (2001)

Shimizu C：“通过 1ck 近端启动子活性的表达可视化小鼠成年胸腺最早亚群中 T 细胞谱系限制的进展”Intern Nutrition。

Takashima A: "Involvement of cyclin dependent kinase5 activator p25 on tau phosphorylation in mouse brain"Neurosci Lett. 306. 37-40 (2001)

Takashima A：“细胞周期蛋白依赖性激酶5 激活剂 p25 对小鼠大脑 tau 磷酸化的参与”Neurosci Lett。

Zhou L: "Two independent pathways of maternal cell transmission to offspring : through placenta during pregnancy and by breast-feeding after birth"Immunology. 101. 570-581 (2000)

周L：“母体细胞向后代传播的两种独立途径：怀孕期间通过胎盘和出生后通过母乳喂养”免疫学。