Autologous somatic cell nuclear transfer in pigs using recipient oocytes and donor cells from the same animal

The objective of the present study was to examine the feasibility of the production of autologous porcine somatic cell nuclear transfer (SCNT) blastocysts using oocytes and donor cells from slaughtered ovaries. Therefore, we attempted to optimize autologous SCNT by examining the effects of electrical fusion conditions and donor cell type on cell fusion and the development of SCNT embryos. Four types of donor cells were used: 1) denuded cumulus cells (DCCs) collected from in vitro-matured (IVM) oocytes; 2) cumulus cells collected from oocytes after 22 h of IVM and cultured for 18 h (CCCs); 3) follicular cells obtained from follicular contents and cultured for 40 h (CFCs); and 4) adult skin fibroblasts. The DCCs showed a significantly (p < 0.01) lower rate of fusion than the CCCs when two pulses of 170 V/mm DC were applied for 50 µsec (19 ± 2% vs. 77 ± 3%). The rate of DCC fusion with oocytes was increased by the application of two DC pulses of 190 V/mm for 30 µsec, although this was still lower than the rate of fusion in the CCCs (33 ± 1% vs. 80 ± 2%). The rates of cleavage (57 ± 5%) and blastocyst formation (1 ± 1%) in the DCC-derived embryos did not differ from those (55 ± 6% and 3 ± 1%, respectively) in the CCC-derived SCNT embryos. Autologous SCNT embryos derived from CFCs (5 ± 2%) showed higher levels of blastocyst formation (p < 0.01) than CCC-derived autologous SCNT embryos (1 ± 0%). In conclusion, the results of the present study show that culturing cumulus and follicular cells before SCNT enhances cell fusion with oocytes and that CFCs are superior to CCCs in the production of higher numbers of autologous SCNT blastocysts

Production of cloned sei whale (Balaenoptera borealis) embryos by interspecies somatic cell nuclear transfer using enucleated pig oocytes

In this study, we examined the feasibility of using subzonal cell injection with electrofusion for interspecies somatic cell nuclear transfer (iSCNT) to produce sei whale embryos and to improve their developmental capacity by investigating the effect of osmolarity and macromolecules in the culture medium on the in vitro developmental capacity. Hybrid embryos produced by the electrofusion of fetal whale fibroblasts with enucleated porcine oocytes were cultured in modified porcine zygote medium-3 to examine the effects of osmolarity and fetal serum on their in vitro developmental capacity. More than 66% of the whale somatic cells successfully fused with the porcine oocytes following electrofusion. A portion (60~81%) of the iSCNT whale embryos developed to the two- to four-cell stages, but no embryos were able to reach the blastocyst stage. This developmental arrest was not overcome by increasing the osmolarity of the medium to 360 mOsm or by the addition of fetal bovine or fetal whale serum. Our results demonstrate that sei whale-porcine hybrid embryos may be produced by SCNT using subzonal injection and electrofusion. The pig oocytes partly supported the remodeling and reprogramming of the sei whale somatic cell nuclei, but they were unable to support the development of iSCNT whale embryos to the blastocyst stage

Porcine Endogenous Retrovirus Transmission Characteristics of Galactose α1-3 Galactose-Deficient Pig Cells

Galactose α1-3 galactose (Gal) trisaccharides are present on the surface of wild-type pig cells, as well as on viruses particles produced from such cells. The recognition of Gal sugars by natural anti-Gal antibodies (NAb) in human and Old World primate serum can cause the lysis of the particles via complement-dependent mechanisms and has therefore been proposed as an important antiviral mechanism. Recently, pigs have been generated that possess disrupted galactosyl-transferase (GGTA1) genes. The cells of these pigs do not express Gal sugars on their surface, i.e., are Gal null. Concerns have been raised that the risk of virus transmission from such pigs may be increased due to the absence of the Gal sugars. We investigated the sensitivity of porcine endogenous retrovirus (PERV) produced from Gal-null and Gal-positive pig cells to inactivation by purified NAb and human serum. PERV produced in Gal-null pig cells was resistant to inactivation by either NAb or human serum. In contrast, although Gal-positive PERV particles were sensitive to inactivation by NAb and human serum, they required markedly higher concentrations of NAb for inactivation compared to the Gal-positive cells from which they were produced. Complete inactivation of Gal-positive PERV particles was not achievable despite the use of high levels of NAb, indicating that NAb-mediated inactivation of cell-free PERV particles is an inefficient process