Generation Of Chimeric Minipigs By Aggregating The Embryos Derived From Somatic Cell Nuclear Transfer

As the body size and physiological structure are similar with human, the pig, especially miniature pig is considered an ideal animal model of human diseases with unique advantages in surgery and xenotransplantation studies. Embryo complementation has become the most important technique of generating chimeric organs in porcine organ-deficient embryos. So far, somatic cell nuclear transfer (SCNT) is regarded as the most reliable approach to produce targeted genetic modifications embryo. The aim of this study was to develop an efficient system to generate chimeric minipigs by aggregating the whole embryos derived from SCNT.1. Primo Vision Time-Lapse were used to observe the development of porcine embryos. The statistics of cleavage time of embryos from SCNT and parthenogenesis was made respectively. The results showed that the cleavage time of 2-,4-,8-,16-cell embryos was 18.5±3.5 h,35±4 h,67.5±3.5 h,80±4.5 h, respectively. There was no significant difference in the cleavage time between the SCNT and parthenogenic embryos, but the time of blastocyst formation of SCNT embryos was longer than the time of parthenogenetic embryo. We also compared the developmental capacity of SCNT embryos in two culture system between the Well of the Well (WOW) system and drop. There was no significant difference in the cleavege rate between WOWs and drop culture system. However, the drop showed a significantly (P<0.05) higher blastocyst rate than the WOW system.2. Synchronous SCNT embryos at different stage were selected for aggregation. The obtained rate of 4-8 cell embryos was 49.5% (657/1326). The obtained rate of 8-16 cell embryos was only 33.9% (499/1474). The rate of aggregation of 4-8 cell embryos was significantly higher than the rate of 8-16 cell embryos (23.9%vs.20.0%). However, there were no significant difference between the aggregate blastocyst and chimeric blastocyst rate of 4-8 cell embryos and the rate of 8-16 cell embryos (54.1% vs.56.0%; 38.8% vs.37.5%). Then the chimeric minipigs were generated by aggregating EGFP-tagged embryos with non-transgenic embryos at 4-8 cell stage. Blastocysts were formed in 50.6% (391/772) of the aggregates. A total of 387 blastocysts were transferred into five recipient gilts of which four gilts became pregnant. The delivery rate of chimeras was at least 11.1% (2/18). Through the tracking of EGFP and the microsatellite examination, skin, heart and kidney were proved to present mosaicism, while spinal cord, spleen, stomach, pancreas, intestine, muscle, lung and liver had no mosaicism.