Expression And Function Of Prdm14 In Porcine Early Embryo

The early embryogenesis is the progress from the zygotes to embryonic tissues stretch without establishing contact with the uterus. During the early embryonic development, a variety of genes begin to express while regulated by a regulatory networks, such as DNA methylation and histone modifications and other associated epigenetic modifications. It will also be regulated after transcription by other factors like autophagy and Micro RNA.PR domain-containing transcriptional regulators 14(PRDM14) is a member of PRDM family, which can promote or inhibit the expression of genes. It has been shown that Prdm14 gene could express from the early embryo to the blastocyst. After the blastocyst stage, Prdm14 gene is specifically expressed in primordial germ cells(PGC). Prdm14 gene in mice and the human exerted the important role in the regulation of gene expression, maintaining pluripotency, and epigenetic reprogramming. Especially, PRDM14 could combine with Dnmt3 a and Dnmt3 b to inhibite their functions, maintain the DNA hypomethylation state and promote the expression of genes. Therefore, we determined to carry out the research on Prdm14 gene.The pig is one of the main livestock products in China. It is widely used to study the organ transplants and animal disease models, because the size of organs and physiological characteristics is similar to humans.Therefore, we selected pigs to explore the expression of Prdm14 genes in embryonic and newborn piglets, and its effect on embryonic development using PCR, q PCR and RNAi technologies.The main results of this experiment are as follows:1.We design three pairs of primers at different sites according to the predicted m RNA sequence of porcine Prdm14 gene on the web of NCBI,then amplify porcine Prdm14 gene by the polymerase chain reaction(PCR) technique. After getting the product with the similar size to the predicted 321 bp, 241 bp and 479 bp, we compared the sequences of products with the predicted sequence. The results showed that the similarity is 98% to 100%. Through comparison of PRDM14 protein amino acid sequences with a variety of common species, the porcine PRDM14 protein is similar to bovine to 88%, and is similar to human to 81%. It is highly conserved in zinc finger region, so the protein is predicted to have similar function.2.We detected the expression of Prdm14 genes in Mâ…¡oocyte, embryonic 2-cells, 4-cells, 8-cells and blastocyst, as well as the heart, liver, spleen, lung, kidney, ovary and testis of new born piglets by using the q PCR. The results showed that the expression level of Prdm14 is highest in Mâ…¡oocytes and reduced in the 2-cells embryos to the lowest. Following the development, the expression of Prdm14 was increased gradually to 8-cell embryos and reduced in blastocysts. Among the main organs of newborn piglets, Prdm14 gene is expressed higher in testis and ovaries, and lower in other organs. PRDM14 protein can be found in each period of embryos development by immunofluorescence staining.3.We interfere the expression of Prdm14 genes by injecting the small interfering RNA into cytoplasmic of early embryos to investgate the function of Prdm14 genes in embryonic development and regulation of gene expression. The results show that after the interference of Prdm14 gene expression, the cleavage rate and blastocyst rate decreased significantly, but there is no significant difference in total cell number of blastocysts and apoptosis.4.We investgate the effect of interference Prdm14 gene on pluripotency genes and apoptosis genes by using q PCR. The results showed that, after interfereing the expression of Prdm14 gene at 4-cell stage, the expression levels of pluripotency genes Oct4 and Sox2 and anti-apoptotic genes Bcl2 are significantly reduced, but there is no significant difference in the amount of expression of pro-apoptotic genes Bax. In the blastocyst stage, there was no significant difference in expression levels of pluripotency and apoptosis related genes.5.We investgate the effect of interference Prdm14 gene on DNA methylation transferases(DNMTs) genes by using q PCR. The results showed that after interfereing the expression of Prdm14 gene at 4-cell stage, the expression levels of Dnmt1 genes are significantly reduced, but no significant difference in the amount of expression of Dnmt 3a and Dnmt 3b. In the blastocyst stage, there was no significant difference in expression levels of these three genes compared to the control group.