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.