The use of reproduction technology for the establishment of transgenic chicken

我们试图通过将逆转录病毒载体感染到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。为此，从2.5天的胚胎中分离出血液循环原始生殖细胞，并用抗SSEA-1抗体标记的磁珠纯化。在体外感染逆转录病毒载体后，将原始生殖细胞移植到2.5d供体胚胎的血管内，其中原始生殖细胞在移植前被部分去除。在11个试验中，共孵化出66枚胚胎，8只萨门公鸡的载体拷贝数为0-0.012。为了克服基因沉默，实现目的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最小启动子组成的复合启动子。我们在逆转录病毒载体上依次克隆了人促红细胞生成素基因、核糖体内部结合位点和tet激活子基因，并将该病毒载体导入鸡胚胎。在所建立的转基因鸡中，成功地在蛋清中表达了促红细胞生成素，但其蛋白产量低于鸡肌动蛋白启动子。

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.