PRONUCLEAR VISIBILITY, DEVELOPMENT AND TRANSGENE EXPRESSION IN IVM IVF-DERIVED PORCINE EMBRYOS/

A total of 1550 zygotes was used to assess the timing of pronuclear vi sibility, embryo development following DNA microinjection, and transge ne expression in IVM/IVF-generated porcine embryos. After centrifugati on, pronuclei could be seen in 61.6% of zygotes. In 55.3% of these onl y 1 pronucleus was visible. Pronuclear visibility was highest at 20 h post-insemination. Zygotes were microinjected with 1 of 2 LacZ gene co nstructs driven by either the SV40 early promoter (pSVON) or the human cytoplasmic beta actin promoter (pbActinLacZ). Development and transg ene expression were assessed after either 48 h or 7 d in culture. Afte r 48 h, significantly more zygotes with a single visible pronucleus de veloped to the 8-cell stage than zygotes in which no pronucleus had be en seen (43.0 vs 24.8%), while those with 2 pronuclei were intermediat e (31.4%). After 7 d, no difference in development to the morula stage was observed between noninjected control embryos (25.5%) and embryos with 1 (21.0%) or 2 pronuclei (22.5%); however, the proportion of embr yos reaching the morula stage in the nonpronuclear group was significa ntly reduced (9.1%). After 48 h in culture, transgene expression was s ignificantly higher in embryos with 2 pronuclei at the time of injecti on than in those with 1 (36.4 vs 17.9%). Alter 7 d in culture, 41.5% o f morulae derived from zygotes with 2 pronuclei and 29.97% of thsoe de rived from zygotes with 1 pronucleus showed signs of transgene express ion. At this stage, significantly more morulae expressed the pbActinLa cZ than the pSVON transgene (43.8 vs 25.8%). More than 80% of putative transgenic morulae or blastocysts showed evidence of mosaicism. These results demonstrate that IVM/IVF porcine embryos are able to develop in culture and express a microinjected transgene.