Here we report the productions of genetically modified cloned Massachusetts General Hospital miniature pig (MGH minipig) using freshly thawed donor cells equilibrated with roscovitine. Fibroblasts were isolated from the ear skin of a 10-day-old male MGH minipig. The donor cells were divided into two groups: cultured for 3 days (culture) and freshly thawed with 500 nM roscovitine. The viability of the donor cells was significantly higher at 0 h (94.6±3.5) compared with 1 h (81.7±5.7) after thawing (p=0.028). After 1 hr of equilibration time, the proportion of G0/G1 stage in roscovitine group was not different from 0 hr group, but not in culture medium group (p<0.01), respectively. Although the developmental characteristics were not different in both methods, the pregnancy and delivery rate in freshly thawed group were significantly higher than that of culture group (p<0.01), respectively. In total, 12 TG cloned MGH minipigs were delivered and the individual cloning efficiency was from 0 to 2.54%. Taken together, the use of freshly thawed donor cells equilibrated with roscovitine may be helpful method to increase the productivity of the genetically modified cloned MGH minipigs.

Pig parthenotes were able to develop in vivo for 30 days with normal morphology. In pig, during blastocyst elongation between day 10 and 12 of gestation, estrogen production and secretion by conceptus increases, serving not only as the signal for maternal recognition of pregnancy, but also as a stimulus for the production of proteins and growth factors within the uterine environment that initiate implantation. Cloning efficiency is still very low regardless of species. To increase the productive efficiency of (transgenic, TG) clones, an advanced somatic cell nuclear transfer (SCNT) method may need. Here we report the productions of transgenic cloned pigs using cloned embryos and parthenotes simultaneously. Fibroblasts were isolated from an ear skin of a 10‐day‐old NIH miniature pig. The ear fibroblast cells were transfected with the alpha1,3‐ Galactosyltransferase knock‐out/human CD46 knock‐in (GalT KO/hCD46 KI). For SCNT, the TG somatic cells were used as donor cells. Immediately after fusion confirmation, the TG cloned embryos and parthenotes were transferred into both oviducts of surrogates. The mean number of TG cloned embryos and parthenotes was 137 (±15.2) and 123(±27.1), respectively. The pregnancy and delivery rate was (55.6%, 10/ 18) (44.4%, 8/18), respectively. Totally 19 GalT KO/hCD46 KI cloned piglets were delivered. Among them, 11 piglets were survived and 8 piglets were born stillbirth. The healthy 5 piglets are still survived.

One of the reasons to causing blood coagulation in the tissue of xenografted organs was known to incompatibility of the blood coagulation and anti-coagulation regulatory system between TG pigs and primates. Thus, overexpression of human CD73 (hCD73) in the pig endothelial cells is considered as a method to reduce coagulopathy after pig-to-non-humanprimate xenotransplantation. This study was performed to produce and breed transgenic pigs expressing hCD73 for the studies immune rejection responses and could provide a successful application of xenotransplantation. The transgenic cells were constructed an hCD73 expression vector under control porcine Icam2 promoter (pIcam2-hCD73) and established donor cell lines expressing hCD73. The numbers of transferred reconstructed embryos were 127 ± 18.9. The pregnancy and delivery rate of surrogates were 8/18 (44%) and 3/18 (16%). The total number of delivered cloned pigs were 10 (2 alive, 7 mummy, and 1 died after birth). Among them, three live hCD73-pigs were successfully delivered by Caesarean section, but one was dead after birth. The two hCD73 TG cloned pigs had normal reproductive ability. They mated with wild type (WT) MGH (Massachusetts General Hospital) female sows and produced totally 16 piglets. Among them, 5 piglets were identified as hCD73 TG pigs. In conclusion, we successfully generated the hCD73 transgenic cloned pigs and produced their litters by natural mating. It can be possible to use a mate for the production of multiple transgenic pigs such as α-1,3-galactosyltransferase knock-out /hCD46 for xenotransplantation.