Antibody Preparation Of SOX2 And Induced Reprogramming Of Porcine Somatic Cells

Embryonic stem cell (ESC) is derived from cells of early embryo and in vitro culturing of these cells can finally maintain the characteristics of self-renewal and pluripotency on development. Understanding the system of transcription control to ESC is the foundation on understanding the process of human development and measuring cell therapy. The Sox2 gene is a transcription factor which belongs to the Sry family. By activating a lot of gene expression with OCT4 in ESC, SOX2 plays an essential role in maintaining self-renewal and pluripotency of ESC. At present, there are only few reports of Sox2 gene expressed in prokaryotic cells and preparation of antibody. In our lab, we have the vector which contains the whole CDS of mouse Sox2 gene. In this research, we subcloned mouse Sox2 gene by PCR, then the prokaryotic expression vector contained the target gene of Sox2 gene was constructed. The Escherichia coli BL21 (DE3) are used as a host for expression. After induced by IPTG, theSox2 protein was expressed with a fusion GST tag. Meanwhile, the purified Sox2 protein acted as immuneogen to be used for polyclonal antibody preparation, which offers powerful bases for the research of its orientation in ES cells and the mechanism of its function.Owing to its pluripotency and self-renewal ability, ESC has been a popular research subject for a long time. Induced Pluripotent Stem Cell (iPSC), as a sister of ESC, is developing speedy. The hundreds of publications show that people already explored quite a few of the connotation and mechanism on human ESC and mouse ESC. As other ungulates, porcine ESC or porcine iPSC faces many obstacles, though they are significant for the research on Animal Modeling and Organ Transplantation. In this research, we tried to reprogram porcine bone marrow mesenchymal stem cell into a pluripotent statement. A vector which contains Oct4, Sox2, Klf4 and cMyc genes is used to express exogenous transcriptional factors. This vector has a expression system that allows us to control the expression levels of factors strictly. As the inducing factors being expressed, with the help from the extracellular factors, the cells were reprogrammed. We isolated total RNA from these reprogrammed cells and analyzed the expressions of the genes by RT-PCR and immunostaining. The results showed that these cells expressed some pluripotency marker. Our findings provide useful references and enlightenments to the prospective porcine ESC research and the reprogrammed cells may bring appropriate material or tools to further the progress on porcine ESC signaling pathways and gene targeting.