Establishment Of A Pluripotent Related Gene Overexpressing Cell Line In Buffalo Fetal Skin Fibroblast

Induced pluripotent stem cells(i PSCs)have the differentiation potential and proliferation ability similar to ESCs,so they have good application prospects in the establishment of disease models and mechanism research,cell therapy,drug discovery and evaluation,transgenic animal production,animal breeding,etc.There are many ways to obtain i PSCs,and the introduction of foreign transcription factors is the most classic induction method.By transferring into the four classic reprogramming factors of OCT4,C-MYC,KLF4 and SOX2,i PSCs have been obtained on a variety of animals.This study combined with SOE PCR and homologous recombination method to construct 2A peptide-mediated Buffalo OCT4,C-MYC,KLF4 and SOX2 four gene co-expression vector PB_OMKS based on Piggy Bac transposon overexpression system.We transfected buffalo fetal skin fibroblasts(BFSF)with this vector and established BFSF_OMKS cell line by drug screening,which laid the foundation for further exploring the related mechanism of buffalo somatic cell reprogramming.In this study,the transcription level of BFSF OMKS cells obtained was analyzed by combining RNA-SEQ technology and bioinformatics analysis,and the effect of overexpressed OCT4 c-MYC KLF4 and SOX2 genes on the signaling pathways related to somatic reprogramming in buffalo was preliminarily discussed.1.Construction of PB_OMKS overexpression vectorIn this study,OCT4,P2 A,C-MYC,E2 A fragments and KLF4,F2 A,SOX2 fragments carrying homology arms were amplified by designing primers carrying homologous fragments,and then they were spliced into OPME fragment and KFS fragment by SOE PCR technology.Finally,homologous recombination technology was used to connect the OPME fragment and the KFS fragment to the same Piggy Bac transposon plasmid carrying the puromycin resistance gene.And PCR identification,plasmid electrophoresis detection and DNA sequencing were used to confirm that the obtained overexpression vector's sequence information was correct.2.Establishment of BFSF_OMKS cell line and authenticationAfter co-transfecting the constructed PB_OMKS plasmid and the transposase expression plasmid into BFSF cells,puromycin was used for cell screening,and finally the purified BFSF_OMKS cell line was successfully obtained.Identification by q RT-PCR proved that BFSF_OMKS cells successfully overexpressed exogenous OCT4,C-MYC,KLF4 and SOX2 genes.Compared with BFSF cells,the relative quantitative level of OCT4 gene in BFSF_OMKS cells was up-regulated by an average fold change of 2231.26,and SOX2 gene was up-regulated by an average fold change of 2189.10,the C-MYC gene was up-regulated by an average fold change of 8.46,and the KLF4 gene was up-regulated by an average fold change of 13.11.Cellular immunofluorescence staining confirmed that the cell line expressed pluripotency-related proteins OCT4 and SOX2.3.Detection of the proliferation ability of BFSF_OMKS cellsThe proliferation ability of BFSF_OMKS cells was tested.By drawing and comparing the growth curves of BFSF cells and BFSF_OMKS cells,it was found that the proliferation rate of BFSF_OMKS cells obtained in this study was slightly slower than that of BFSF cells,but it maintained a good proliferation capacity and stable passage ability and could maintain subculture for more than one month.At the same time,the transcriptome data showed that the BFSF_OMKS cell line expressed pro-survival genes PTPN13,CFLAR and TRAF14.Analysis of transcriptional level of BFSF_OMKS cell lineTranscriptome sequencing of BFSF cells and BFSF_OMKS cells revealed that compared to BFSF cells,the LIF,Activin,BMP4 and WNT signals in BFSF_OMKS cells were activated,and the genes related to these signals LIF,INHBB,BMP2 / 4,WNT4 / WNT5 A appeared significantly up-regulated.In addition,BFSF_OMKS expresses embryonic stem cell signal FGF2,mesoderm development-related gene DLX5 and endoderm development-related gene ZFHX3.In order to verify the reliability of these transcriptome data,primers were designed for 20 genes including OCT4,SOX2,LIF,DLX5,FGF2,INHBB,BMP2,BMP4,SMAD9,and SMAD1,and q RT-PCR were performed on sequencing samples.The results are consistent with the transcriptome data,proving that the data obtained by this transcriptome sequencing is highly reliable.The above results indicate that the BFSF_OMKS cell line obtained in this study has initially entered the reprogramming state,and has good proliferation and passaging ability.Therefore,this cell platform can be used to screen small molecule compounds that promote the reprogramming of buffalo somatic cells and investigate the related phenomena and molecular mechanisms of cell reprogramming in buffalo somatic cells reprogramming.