Study On Handmade Somatic Cells Nuclear Transfer Embryos In Pig

This study selected fetal fibroblasts as nuclear donor cells, reconstructedembryos of pigs will be obtained by chemically assisted handmade enucleationmethod. Following that, did some work on hand-made nuclear transfer on pig. Thepurpose of this study were to set up a suitable system of hand-made cloning for ourlaboratory and complete the basic work for the efficiency of porcine somatic cloning.The main results were as follows:1. By using pig ear tissue culture inoculation, cultivation3~4d, It was seen thatthe cell block climbed out of outward along the edge of the organization. There weretypical fetal fibroblasts beside the surrounding tissue in6d, and the cells were frozenby30%FBS+10%DMSO+60%. When cells were recovered and obtained cells ofthe survival rate of90%. Through the digestive process, the pig fetal fibroblast cellswill be obtained to provide the suitable donor cells.2. Inducing the nucleus extrusions on porcine oocytes by the demecolcine. Therewere effection on porcine oocytes of nuclear extrusions of different concentration ofdemecolcine and time of nuclear extrusions. The result showed:(1)Through adding tothe different concentration of demecolcine to treat the porcine oocytes after1h, therehad no significance difference in every groups, while the nucleus extrusions rate ofthe0.4μg/mL groups was the highest (80.8%),(2) Inducing the nucleus extrusions onporcine oocytes by0.4μg/ml concentration of demecolcine, There were differentnucleus extrusions rates, respectively,75.5%,75%,74.55%, that it was taked the30min,40min,50min,60min. There had no significance difference in nucleusextrusions rate of every groups.3. Studying on the effection of the formation of enucleated porcine oocytefusion rate in the different condition of electric field intensity, electric pulse frequencyand the electric field. The result showed:①The rate of enucleated porcine oocyte fusion rate of30μs,1DC,0.8kv/cm group was higher than the other groups, the ratewas94.7%. When the field strength was increased, the formation of enucleatedporcine oocyte fusion rate began to decrease (88.6%,87.7%,84.3%).②The rate ofenucleated porcine oocyte fusion rate was94.7%, when the reaction condition was0.8kv/cm, one times,30μs. The rate of enucleated porcine oocytes fusion were86.5%and83.8%when that was60μs and90μs. The fusion rate will have asignificant decrease.③There had no significant difference in the fusion rate of onetime and two times in0.8kv/cm,30μs (94.7%,95.6%P﹥0.05), but those groups hadhigher the fusion rate of three times groups(81.7%).4. Nuclear transfering the porcine oocytes by traditional nuclear transfer andmanipulated hand-made nuclear transfer methods, The result showed: that there hadno significant difference in the rate of cleavage （53.1%，55%）and blastocyst bytraditional nuclear transfer and manipulated hand-made nuclear transfer.