Establishments Of Buffalo Cell Lines Overexpressing Pluripotent Genes And Preliminary Study On Its Reprogramming State

Recently induced pluripotent stem cells have been widely applied to disease model and biomedical research,but the research on large animal-induced pluripotent stem cells,especially in buffalo,remains to be improved.To further explore the reprogramming mechanism of buffalo fibroblasts and the effects of OCT4(O),SOX2(S),LIN28(L)and NANOG(N)four factors on the buffalo fetal skin fibroblast-reprogramming,we have constructed PLVX＿OL(vector overexpressed OCT4 and LIN28 genes)and PLVX＿SN(vector overexpressed SOX2 and NANOG genes)using lentivirus over-expression vector PLVX＿IRESZs Green based on the self-cleavage of the 2A peptide.Then introduced the two vectors into buffalo fetal skin fibroblasts separately and jointly.And then verified the expression of pluripotent related genes in the three groups of cells in different ways.RNA-seq technology and bioinformatics analysis were used to analyze the different groups of cellsThe results showed that the overexpression of the combination of OCT4,LIN28,SOX2 and NANOG factors in BSF could activate WNT,LIF,MAPK and PI3 K signaling pathway,etc.And OCT4,SOX2,NANOG,LIN28 A,KLF4,FGF2,LIF,LIFR,WNT5 A,WNT11,WNT2 B,BMP4 and FZD3 multipotential factors were significantly up-expressed.Epithelial marker genes such as TJP1(ZO-1)and CTNNA1 were significantly up-regulated,and marker genes of fibroblasts such as SNAI1,CD44,ZEB1 and THY1 were significantly down-regulated,indicating that BSF＿OSLN cells had begun to enter the MET state and were in the early stage of reprogramming.The expression pattern of cells overexpressing OCT4 and LIN28 and cells overexpressing SOX2 and NANOG was more similiar to that of fibroblasts themselves.The above results indicated that the application of the combination of transcription factors of OSLN could obtain a cell line at the early stage of reprogramming.It is expected to further explore the molecular mechanism of buffalo somatic cell-reprogramming by screening small molecular compounds that promote reprogramming,and it will lay a foundation for further exploring the molecular mechanism of buffalo somatic reprogramming and obtaining stable passage of buffalo iPSCs...