The use of reproduction technology for the establishment of transgenic chicken

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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.