抄録
In order to introduce exogenous DNA into early chicken embryos by transfecting the stage X (EG&K) blastoderm, in vivo electroporation was applied to it. A pair of novel electrodes was devised for applying electric pulses vertically to the blastoderm layer. Fertilised eggs at stage X were broken and the blastoderm was placed on top of the yolk. One of the electrodes was placed on the blastoderm layer through the yolk membrane, and the other electrode was placed on the bottom of the yolk. DNA (GFP gene) was injected into the blastoderm, and electric square pulses were applied 5 times at 5-100V for loading periods of 50 msec per pulse at one-second intervals. The manipulated embryos were transferred into host eggshells and incubated for 4 days. The viability of the manipulated embryos was 67.0% (59/88) on day 3 of incubation. The expression pattern of GFP gene was mostly mosaic in the embryos, and the percentages of embryos that expressed the GFP gene were 97.7% (86/88), 93.2% (82/88) and 75.0% (66/88) on days 1, 2 and 3 of incubation. A high rate of GFP gene expression was observed in the embryonic and extra-embryonic tissues (54.2%, 32/59) and also in the extra-embryonic tissues only (27.1%, 16/59) on day 3 of incubation. The expression of the GFP gene throughout the embryonic tissues was also observed. These results suggest that the system for applying electric pulses vertically to the blastoderm layer is effective in introducing exogenous DNA into early chicken embryos.
