| To explore the possibility of generation of transgenic chicken, the Growth Hormone and Human tissue kallikrein 1 as target exogenous genes were used in two methods respectively: by direct microinjection into chicken germinal disc and by means of sperm vectors. The results showed that:1 .The modified windowing technique may be broadly applicable in emerging technologies in avian transgenesis and development . So we sealed the shell windows in fertilized chicken eggs with 8 different shields. The results were as follow: the 5 treated groups sealing materials were (1)eggshell membrane + wrap film (injecting antibiotic for successive 3 days after sealed ) (2) eggshell membrane + wrap film (3) wrap film + cotton (4) eggshell membrane + sealing wax (5) eggshell membrane + eggs shell powdery remnant + sealing wax The survival rates of these 5 treated groups were not significantly different (P>0.05) before the 19th days .The left 3 groups sealing materials were (6) eggshell membrane (7) eggshell membrane + eggshells powdery remnant + egg white (8) eggshell membrane + eggshells powdery remnant. The development of those 3 groups embryos were not good .The hatchability of the first 4 treated groups were 9.6%, 5.1%, 4.4%, 2.4% respectively. Compared with the 8 treatments, the method of eggshell membrane + wrapfilm.is the best choice for production of transgenic chicken in practical applications.2.To explore the possibility of generation of transgenic chickens by direct microinjection of transgenes into the germinal disc of developing embryos, 1.5 kb human growth hormone (hGH) mini gene fused with goat β -lactoglobulin (BLG) gene promoter was mixed with Lipofect Mine reagent and injected into the germinal disc of 24h-developing embryos. Among 81 microinjected embryos, 4chicken were hatched with hatchability of 4.9% (4/81). Both dot blotting and Southern blotting analyses showed that the BLG-hGH construct was integrated into the genomes of 3 out of 44 embryos died during 4 to 18 days of incubation and that of 3 hatched chicks. The exogenous DNA was detected in 12.5%of the embryos or tissue.3. Transmitted the exogenous DNA of human tissue kallikein-1 by direct microinjection into chicken germinal disc .The window was sealed with eggshell membrane + wrap film. Among 239 microinjected 14 chickens were hatched with hatchability of 5.9%( 14/239). We detected the 4-7days and the tissues of 7-20 days of microinjecting embryos. The result of dot blotting was 22.2%. And the P.CR was carried out with these positive samples and the tissue of hatched chicken 3.7% exogenous DNA was detected in embryos and chicken's tissue.According the results of experiments, the genes with different length such as hGH and Klkl can be expressed in the chicken. These data further demonstrated that direct microinjection of transecting exogenous lipofection-DNA can be a useful method for the production transgenic chicken.4.Transgenic chicken were produced by sperm-mediated exogenous DNA transfer method .The sperm were incubated in a lipofection-DNA,and the fertility is normal The results of PCR showed the sperm which incubated in 37℃ with lipofection-DNA carried exogenous DNA Combined with Artification Insemination (AI) technique, and human tissue kallkrein-1 as target genes were used. We defected the 24 chickens, the uncertain positive samples were 79.2%( 19/24) by dot blotting, and the result of PCR is 29.2 %( 7/24).According the result of experiment, the chicken's sperm cells are able to transfer exogenous lipofection-DNA and the sperm- mediated exogenous DNA transfer method can be a useful way of generating transgenic chicken.
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