Preliminary Study Related To The Effect Of Over Expression Of JHDM2A On Reprogramming Effciency Of Porcine IPSCs

iPS technology lays a foundation for the construction of disease models,the development of new drugs,gene therapy and regenerative medicine,which is a milestone in the field of stem cell research.Pig is physically similar to humanity.Therefore,it is of great significance to study porcine iPS cells.At present,the reprogramming efficiency of porcine fibroblast induced to iPS is less than 1%.Histone H3K9 methylation severely blocks iPS induction,while histone H3K9 demethylation promotes iPS induction.The histone methylation level is regulated by histone methyltransferases and histone demethylase.JHDM2A(the JmjC domain histone demethylase 2),which specifically catalyzes the demethylation of H3K9me1/2.It had found that JHDM2A regulated mouse embryonic stem cell fusion-induced reprogramming of adult neural stem cells and regulated self-renewal in embryonic stem cells.In this study,DOX induced lentiviral vector system was used to induce porcine pluripotent stem cells(piPSCs).On this basis,the JHDM2A gene was further overexpressed.Then,we investigated the effect of histone H3K9mel/2 methylation level on the piPSCs induced efficiency,and explored its molecular mechanism.The results laid the foundation for further improving the induced efficiency of iPSCs and establishing the ESCs for big livestocks.The main results of this study were as follows:1.Cloning of JHDM2A gene and construction of its expression vectorAccording to the porcine JHDM2A gene mRNA sequence published on NCBI,we designed the specific amplification primers to clone the porcine JHDM2A gene.It was found that the cloned CDS length was 3945 bp,which encoded 1315 amino acids.The results of multiple amino acid sequence comparison showed that the porcine JHDM2A shared 93.5%,94.7%,94.7%and 93.8%homologous compared with those of Bubalus bubalis,Bos taurus,Ovis aries and Homo sapiens,respectively.Phylogenetic tree analysis indicated that the JHDM2A gene was highly conservative in the evolutionary process.The immunohistochemical results suggested that the JHDM2A protein was expressed in porcine follicle of different development stages.Then,The eukarybtic expression vector of pLVX-IRES-ZsGreenl-JHDM2A was constructed and validated.2.The effects of the overexpression of JHDM2A gene on porcine iPSCs formation and the expression of related genes.Firstly,the OSKM factors was introduced into porcine fibroblast cells to induce iPSCs using DOX inducible lentiviral vector system.On this basis,the JHDM2A gene was overexpressed,and the obtained iPSCs were identified respectively.When the JHDM2A gene was overexpressed,fibroblasts were deformed on the third day,and the piPSCs clone was formed at eighth day in the experimental group,which was one and two days earlier than that of the untreated group.The positive clones were detected by AP staining.Compared with the untreated group,the experimental group which the JHDM2A gene was overexpressed increased by 8 times,and the iPSCs clones were more uniform in size,more flat in edges,denser in cells,and deeper in the coloration of the AP staining.The results of RT-PCR showed that the pluripotency genes of Klf4,c-Myc and Nanog were expressed,Sox2 was weakly expressed in the three kinds of cells detected.Oct4 was expressed in the experimental and untreated group,while weakly expressed in PFF.The results of quantitative PCR showed that the expression of the pluripotency gene Oct4,Sox2,Klf4,c-Myc,Nanog was significantly higher in the experimental group than in the untreated group(P<0.05),and the expression of Oct4 was very significant(P<0.01).After the removal of DOX,the exogenous genes in the experimental group of piPSCs cells were silenced,and the endogenous gene Oct4 was almost no change from generation to generation,Sox2 and Nanog decreased gradually,and Klf4 and c-Myc gene increased first and then decreased.Compared with the untreated group and the PFF group,the expression of the histone methylation gene Tcll was significantly higher(P<0.05),and Tcfcp2ll and Zfp57 were significantly lower in the experimental group(P<0.05).The above results show that the porcine JHDM2A gene is cloned and its eukaryotic lentivirus expression vector is constructed.The overexpression of JHDM2A gene can improve the induced efficiency of piPSCs by regulating histone methylation level.