Production of Cloned Miniature Pigs by Enucleation using the Spindle View System

Abstract

Porcine somatic cell nuclear transfer (NT) has been successfully performed, but its efficiency remains quite low. In this study, we improvised on the enucleation method to enhance the development of NT embryos. Initially, an experiment was performed to determine the location relationship between the metaphase plate and the first polar body, where the results showed that the metaphase plate may frequently be displaced during the varying period of maturation process. When the metaphase plates were removed using the 'blind' enucleation method, the enucleation rate was affected by the maturation time; however, when the spindle view system was used, an enucleation rate of 100% was achieved. In the next experiment, these two methods were used to construct embryos: the fusion efficiency was significantly higher (p < 0.01) in the spindle view system group and the development rates of the reconstructed embryos were significantly higher in the spindle view system group compared with the 'blind' enucleation group (p < 0.01). An average of 174 (141-210) cloned embryos from the spindle view system group were transferred into five surrogate pigs and one piglet was delivered at 114 days after embryo transfer by caesarean section. DNA analysis confirmed that the piglet was genetically identical to the male donor pig. We showed that enucleation by the spindle view system is the another new technique compare the handmade cloning method [Theriogenology 2007: 68, 1104] to promote the development of the reconstructed embryos, and that a full-term cloned pig could be produced using this method.

Developmental Ability of Miniature Pig Embryos Cloned with Mesenchymal Stem Cells

“…In general, due to rapid growth and the potential for multiple cell divisions, fetal fibroblasts have been commonly used as nuclear donors in pig embryo cloning. The blastocyst rates of pig embryos cloned with FFs are below the level of 20% [30][31][32]. These data are substantially in accordance with our data showing that 14.5% of embryos cloned with FFs developed to blastocysts.…”

“…The usefulness of miniature pigs as an animal model has been proven; but nevertheless, only a limited number of studies have reported on cloned miniature pigs derived from somatic donor cells [30,31,[34][35][36]. Because of the comparison with domestic pigs, production of cloned miniature pigs using miniature pigs oocytes has limitations.…”

“…These findings are similar to the observations of the above-mentioned studies. Our group has observed the expression profiles of genes involved in transcription, DNA methylation, histone deacetylation, apoptosis and embryonic growth at different developmental stages, and some genes in embryos cloned with MSCs closely resembled those of in vivo produced embryos [7].The usefulness of miniature pigs as an animal model has been proven; but nevertheless, only a limited number of studies have reported on cloned miniature pigs derived from somatic donor cells [30,31,[34][35][36]. Because of the comparison with domestic pigs, production of cloned miniature pigs using miniature pigs oocytes has limitations.…”

Effects of enucleation method on in vitro and in vivo development rate of cloned pig embryos

“…This approach results in an enucleation rate that varies widely among laboratories (40%-90%) depending on the skill and experience of the operators; it is usually difficult to reach a high enucleation rate due to "blind" localization of the nucleus [2][3][4][5] . To increase enucleation efficiency, spindle imaging system-assisted (SIS-A) enucleation has been used for oocyte enucleation in pig, cattle and other species [6][7][8][9][10][11][12][13] . This method employs a polarized light microscope to visualize the meiotic spindle-containing oocyte nucleus.…”

“…This method employs a polarized light microscope to visualize the meiotic spindle-containing oocyte nucleus. The SIS-A approach can achieve a high oocyte enucleation rate (88%-100%) in several mammalian species [8,10,12,13] and improve the in vitro development efficiency of cloned embryos in pigs and cattle [6,7] . Fluorescence dye staining has also been used to localize clearly the oocyte nucleus before enucleation [14,15] .…”