In Vitro Culture Of Nuclear Donor Cells And Its Effect On The Production Of Cloned Embryos In Swine

Somatic cell nuclear transfer (SCNT) behaved huge potential and prospect in good quality domestic animal breeding, homogeneous experimental animal production, endangered mammalian species protection, therapeutical cloning, as well as transgenic animal production and so forth. Currently, many kinds of somatic cells were used as nuclear donor cells to produce cloned animals. Cloned pigs by SCNT have some advantages potentially to be used as models of human diseases and donor organs for xenotransplantation, and have been the focus in the SCNT area. Although some progress have been made to yield clone piglets with SCNT technique, there still existed many problems, e.g. the total efficiency of clone pig production was still low with less than 1% of transferred embryos survival to the term and the cloned animals may have abnormal development. Donor cell selection is critical for the success of clone animal production, and it is also an important factor impacting the cloning efficiency. The present study was systematically conducted to examine the effects of cell culture, different donor cells, number of passages on the development of nuclear transfer porcine embryos, so as to establish a preliminary procedure for porcine cloning.The porcine embryonic fibroblasts (PEF), porcine cumulus cells (pCC), porcine granulosa cells (pGC) together with porcine ear fibroblasts cells (PFC) used for SCNT were isolated by tissue explant culture and enzymatic digestive culture method. These cells were frozen and rewarmed to investigate the cryopresevation effects of 2 different programs: Method A. Cells were equilibrated at 4℃for 0.5 h, stored at -20℃for 2h and -70℃overnight and then plunged into liquid nitrogen. Method B. Freezing tubes containing resulting cells were put in Nalgene Cryo-freezer kept in a refrigerator at -70℃overnight and then tubes were plunged into liquid nitrogen. Morphological observations of the cells, cell viability by MTT assay, the cell growth curve and analysis of chromosome karyotype of passaged cells were conducted before using them as donor cells for SCNT. Porcine oocytes were collected from ovaries obtained at slaughterhouse and cultured in vitro for 40-44 h, enucleated in manipulation, and then the donor cells including PEF, pCC, pGC or PFC were transferred into enucleated oocytes by microinjection to reconstruct new embryos. Reconstructed embryos were cultured for 7 days to evaluate their cleavage rate and further development. The results showed as follows.1. The survival percentage and attachment percentage obtained by method B is remarkably higher than method A in the cryoperation of all the four kinds of somatic donor cells. So method B is confirmed to be the best scheme for freezing the somatic cells in this experiment.2. The grwoth curve of cultured somatic cells in vitro appeared "S" shape and illustrated that cells growing went through three phase as latent phase, logarithmic phase and stagnate phase. It showed that the cells would come into lag phase during the first day of culture, after this the cells would come into logarithm growth phase which would last for two or three days, and finally they would come into plateau phase which would last for three days, then the amount of the cells would descend, because the cells began to fell off and died gradually.3. The chromosome examination showed that PEF in feasible culture system could retain normal chromosome of double character at least 8th passage, pCC could retain normal chromosome of double character at least 5th passage, pGC could retain normal chromosome of double character at least 5th passage and PFC could retain normal chromosome of double character at least 6th passage. So these cells could be used as donor cell for nuclear transfer.4. In this nuclear transfer experiment, we used PEF, pCC, pGC and PFC as donor cells in order to compare their effects on SCNT. The results showed that all these 4 groups could yield clone morulae or blastocysts, and the cleavage rate of PEF (70.2%), pGC (73.3%) and pCC (82.5%) are significantly higher than that of PFC (61.2%, P<0.05), the morula or blastocyst formation rate of pGC(39.6%) and pCC (47.6%) were significantly higher than that of PFC (28.4%, P<0.05).5. Effects of different passages of PEF, pGC and PFC on SCNT were investigated and the results showed that: the cleavage rate of the 2nd passage (P2) of PEF (79.0%) was significantly higher than the 3rd passage (58.1%, P<0.05), but the morula or blastocyst formation rate of between P2 and P3 showed no significant difference; the cleavage rate of P3 of pGC was significantly lower than the other passages, but the morula or blastocyst formation rate of P2 of pGC was significantly higher than the other passages (P<0.05); the cleavage rate of different passages of PFC showed no significant difference, but the morula or blastocyst formation rate of P0 (36.8%) and P3 (44.1%) were significantly higher than Pl(10.5%) and P2(13.5%, P<0.05).6. There were 2 different ways to get pCC used as donor cells in this experiment: directly isolation and passage, to examine their effects on SCNT. The cleavage rate of the cumulus cells isolated by different ways showed no difference, but the morula or blastocyst formation rate of the cumulus cells which were directly isolation from COC (47.6%) were significantly higher than that from passaging cumulus cells (22.2%, P<0.05). Therefore, the cumulus cells directly isolated from COC were able to be used as nuclear donor cells.