The Construction Of Pig Histones,Histone Variant Expression Vectors And The Effects On Pig Somatic Cell Reprogramming

Under normal physiological conditions,protein post-translational modifications regulate the process of signal transductions,activities of enzymes,and the process of protein localization,recognition and degradation.Histone protein post-translational modifications influence the interactions between DNA and binding proteins,which alerts DNA duplication and transcription process.The modifications of histones further regulate the chromatin structures.After post-translational modifications,histone transformed into hisone variants,which were widely expressed in various cells.As the corresponding variants of histone H2 A and H2 B,TH2A and TH2 B,are encoded by Hist1h2 aa and Hist1h2 ba,respectively and specifically expressed in the process of spermatogenesis and oogenesis.TH2 A and TH2 B facilitate the activation of paternal genes after fertilization and embryonic reprogramming.Our research aimed to construct the transfection vectors of H2 A,H2B,TH2 A and TH2 B and analysis the effect of H2 A,H2B,TH2 A and TH2 B overexpression on the reprogramming efficiencies of Pig fetal fibroblast cells.Firstly,the whole coding region of H2 a,H2b,Th2 a and Th2 b were amplified by PCR with total RNA of Pig oocytes extracted and cDNA reversed.The genes were then cloned into p GEM-T vectors and confirmed by commercial sequencing,respectively.Each correct gene were cloned into p EGFP-N2 and pCMV-Red vectors to establish transfection vectors,respectively.The various combinations of transfection vectors were then transfected into Pig fetal fibroblast cells with electroporation followed by G418 selection and cell reprogramming to analysis the effect of hisones and histone variants on the Pig fibroblasts reprogramming and optimize the combinations of transfection vectors.Finally,according to the changes of cell morphology and specific genes expression levels,we concluded that overexpression of Th2 a and Th2 b facilitated the process of mesoepithelial transformation epithelium(MET)during reprogramming.In summary,Pig histone H2 A,H2B and histone variant TH2 A,TH2B were successfully constructed and transfected into Pig fetal fibroblast cells.And our results showed that histone variants promoted the reprogramming process of Pig fetal fibroblast cells.And we will further investigate the mechanism of histone variants during the induction of pig pluripotent stem cells.