Tracking Of Cellular Reprogramming In Chicken Somatice Cells By PouV Gene Reporter

Cell reprogramming technology can reverse differentiated somatic cells into Induced Pluripotent Stem Cells(iPSC)and can differentiate into any cell type,which is helpful for preservation of endangered species and developmental biology using avian model.In this study,a fluorescence gene was inserted into the last exon of PouV gene in chicken cells for use as reproter to track the somatic cell reprogramming process,thereby facilitating the research of cellular reprogramming and its underlined mechanisms.In the first experiment,we successfully inserted the red fluorescent gene mCherry into the last exon of chicken PouV gene by the Homology-Mediated End Joining(HMEJ)mediated by CRISPR/Cas9 technology.The expression of PouV-mCherry expression will be driven by chicken endogenous PouV promoter and could be used as a reporter to illustrated the pluripotency of the cells during reprogramming,which will facilitate the derivation of iPSC from somatic cells.Secondly,the PiggyBac transposon was used to transfer the pluripotent transcription factors Nanog,PouV and Lin28(NPL)into pPouV-mCherry somatic cells to induce pluripotency in these cells.The overexpression vector PB-NPL-EGFP carrying chicken Nanog,PouV and Lin28(NPL)expression cassettes was successfully constructed and transfected into pPouV-mCherry chicken embryonic fibroblasts,cardiomyocytes and DF-1 cells.Interestingly,chicken embryonic fibroblasts,cardiomyocytes underwent apoptosis during after transfection,while red DF-1 cells were observed 7th day of induction and large red clonies appeared on the 30th day of induction.The derived colonies demonstrated clear boundary and stereoscopic morphology,similar to iPS or ES cells.The cells could be maintained up to 40 days but began to undergo apoptosis after subculture.Charaterization of these cell colonies showed that they were positive for alkaline phosphatase.And pluripotent transcription factors Nanog,PouV,Lin28,c-Myc and Klf4 were significantly up-regulated compared to their parent somatic cells.Stem cell-related and germ cell-related genes TBX3,TFCP2L1,ENS-1 and c-Kit were also significantly upregulated in these derived cells.The results indicated that the cells in red colonied had been subjected to cellular reprogramming and demonstrate the potential of pluripotency 30 days of induction.In summary,the endogeneous PouV gene in chicken cells was targeted and labeled with red fluorescent by using CRISPR/Cas9 technology.It could be used as a reporter to track the process of cellular reprogramming,which will facilitate the research in chicken iPSC derivation and its underlined mechanism.