The use of reproduction technology for the establishment of transgenic chicken

利用繁殖技术建立转基因鸡

基本信息

批准号：
18360393
负责人：
IIJIMA Shinji
金额：
$ 11.35万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

We were trying to establish transgenic chicken by infecting a retoroviral vector to purified primodial germ cells and successive grafting of cells to donor embryos. For these, blood-circulating primodial germ cells were isolated from day 2.5 embryos, and purified by anti-SSEA-1 antibody-conjugated magnet beads. After in vitro infection of a retroviral vector, primodial germ cells were grafted to blood vessel of day 2.5 donor embryos, of which primodial germ cells were partially removed just before the grafting. In total 11 trials, 66 embryos were hatched and the vector copy number of male chickens was determined to be 0-0,012 in samen (8 individuals) . Now, we are trying to establish transgenic chicken by mating of these chickens.To overcome gene silencing and attain egg-white specific deposition of target protein, we used a hybrid promotor composed of ovalbumin enhancer, tet binding site and CMV minimum promoter. We cloned human erythropoietin gene, internal ribosome binding site and tet activator gene by this order in a retroviral vector and introduced the viral vector to chicken embryos. In the established transgenic chikens, erythropoietin was successfully produced in egg-white but the amount of the protein was less than that obtained by chicken actin promoter.

我们试图通过将逆转录病毒载体感染纯化的原始生殖细胞并将细胞连续移植到供体胚胎来建立转基因鸡。对于这些细胞，从第 2.5 天的胚胎中分离出血液循环的原始生殖细胞，并通过抗 SSEA-1 抗体缀合的磁珠进行纯化。体外感染逆转录病毒载体后，将原始生殖细胞移植到第2.5天供体胚胎的血管中，其中在移植前部分除去原始生殖细胞。在总共11次试验中，孵化了66个胚胎，并且确定雄性鸡的载体拷贝数在samen（8个体）中为0-0,012。现在，我们正在尝试通过这些鸡的交配来建立转基因鸡。为了克服基因沉默并实现目标蛋白的蛋清特异性沉积，我们使用了由卵清蛋白增强子、tet结合位点和CMV最小启动子组成的混合启动子。我们按照逆转录病毒载体的顺序克隆了人促红细胞生成素基因、内部核糖体结合位点和tet激活基因，并将该病毒载体导入鸡胚胎。在已建立的转基因鸡中，在蛋清中成功产生了促红细胞生成素，但蛋白质的量少于鸡肌动蛋白启动子所获得的蛋白质的量。