139 EFFICIENT CONDITIONS OF CYTOPLASMIC MICROINJECTION OF FOREIGN DNA TO GENERATE PORCINE TRANSGENIC EMBRYOS

Abstract

To establish the efficient cytoplasmic microinjection system in the porcine embryos, pEGFP-N1 plasmid were microinjected into porcine parthenogenetic (PA) and in vitro-fertilized (IVF) embryos to investigate the optimal injection time, volume, and concentration. In experiment 1, to investigate the optimal injection time, development rates were compared among groups of 4 different time durations (2, 4, 6, and 8 hours) in the PA and IVF embryos with time point after activation and sperm removal, respectively. There were no significant differences (P < 0.05) between the 4 groups regarding the cleavage rates. However, there were significant differences (P < 0.05) in development to the blastocysts (4.4, 8.9, 3.9, 0.6%) and GFP expression in blastocysts (1.3, 5.7, 2.3, 0.0%), which was injected after postactivation of 4 hours compared with another 3 groups. The IVF embryos injected after 2 and 4 hours expressed GFP significantly higher than the other two groups, which injected at 6 and 8 hours (P < 0.05). In experiment 2, EGFP-N1 with 2 different concentrations (20 and 50 ng μL^–1) was injected in the PA and IVF embryos to investigate the optimal concentration. In PA embryos, there were significant differences in 20 ng μL^–1-injected embryos, which had higher cleavage (58.8 v. 41.9%) than blastocysts (13.0 v. 11.1%) and GFP expression rates (P < 0.05). In IVF embryos, GFP were expressed only in 20 ng μL^–1 embryos, GFP (4.2%) in the blastocysts showed no significant difference in the cleavage (77.3 v. 64.7%) and blastocyst rates (26.4 v. 23.5%). In experiment 3, three different volumes (5, 10, and 20 pL) were microinjected into porcine embryos to determine the most appropriate volume. Out of 3 groups, significantly higher development rates of cleavage (68.3, 58.0, 29.3%), blastocysts (11.7, 12.7, 0.5%), and GFP-expressed blastocysts (2.9, 7.8, 0.0%) were shown in the 10-pL group (P < 0.05). In conclusion, these results imply that a 20 ng μL^–1 concentration, 10 pL of volume, injection 4 hours after activation for PA embryos, as well as injection 2 and 4 hours after sperm removal, a 20 ng μL^–1 concentration, and 10 pL of volume for IVF embryos were more effective cytoplasmic microinjection conditions.