Reproduction Of Cloned Pigs And Expression Analysis Of Imprinted Gene

The somatic cell nuclear transfer of pigs is injecting donor cells into enucleated oocytes,make the donor nucleus carrying on normal reprogramming through the auxiliary activation, then transplant the embryo into acceptor animal,which process of cloned offspring. This technology has been widely used, but its success rate is very low, thus wasting a lot of manpower, material resources. At present, the best method is optimization of Culture Conditions for the nuclear transfer process, for example,changing the composition of culture media.Our experiment research other factors involved in the process, in order to improve the success rate of nuclear transfer.Imprinted genes are a class of genes plays an important role in mammals, they are exclusively expressed from the one part of parental alleles, and did not express from the other part of parental alleles.If anyone of these two alleles have be abnormality,it will lead to the abnormal expression of imprinted genes and functional disorders. Except for somatic cell reprogramming developmental failure could cause abnormalities of cloned animals, the in vitro procedure and culture conditions also impact on development. And cause these abnormality have been appearanced mostly because all imprinted genes have been damaged,and could not be expressed normally. Therefore,reserach some of the imprinted gene expression differences between normal pigs and cloning pigs could help us better understanding of the changes of imprinted genes in nuclear transfer process, and optimization the system of clone process..The experiment consists of two parts. The first part is preparation of cloned pigs:comparison of oocyte maturation rate in different seasons, and achievement ratio from different operators. It contain fusion rate, cleavage rate and blastocyst rate of reconstructed embryos after the injection, which were compared to some details of the injection process to increase the success rate of clone. By comparison of the data, the maturation rate of oocyte and embryo development in spring should be significantly better than in winter, therefore, the best season to carry on the cloned pig transplant experiments is in spring. In the process of injection,take moderate cytoplasm near the corpus polare,because taking too little could not guarantee complete removal the genetic material of the oocyte, taking too mush would lead the reconstructed embryo to die or maldevelopment; The injection speed should be rised on the basis of quality assurance as possible, so as to avoid the oocyte outside for too long time.The second part is researching for the expression of imprinted gene. The cloned pigs were dead in two days after born. In order to explaned the abnormal phenotype of cloned pig, we collected the heart, liver, spleen, lung and kidney of the dead pig, and normal pig’s organ were also collected to be control. It was analysed that the different of Peg10, H19and Phlda2gene expression level between the cloned pig and normal hilt different organ. It was expected that to explaned the reason of abnormal cloned pig by using these molecular mechanism. The experimental results show that the expression of PEG10gene in kidney of cloned pig was significantly higher than the expression level in normal pigs. All expression level of imprinted gene in cloned pigs have difference from normal pigs.However, due to individual differences, the difference of expression of imprinted genes were not significantly. Is this experiment, we have validated the functions of the two imprinted genes that named PEG10and PHLDA2. PEG10could promote fetal development, and the over expression of PHLDA2would restrict fetal growth,and lead to abnormal development.