Study Of Somatic Cells Reprogramming By The Different Methods

Animal embryonic stem cells(embryonic stem cells,ESCs) provide a powerful tool forstudies of cloning, early embryonic development, gene targeting and regenerative medicine.However, because of the difficulties on obtaining materials and culturing and other reasons,the research of large animals ES cells has beening facing many barriers. At present, it hadfound that by using exogenous pluripotent transcription factors, somatic cells couldreprogramme to a pluripotent state, and the cells were called induced pluripotent stem cells(iPSCs). iPS cells could avoid the difficulties caused by ES cells, and replace ES cells aspluripotent stem cells. Thus, iPS cells have become a research hotspot. Nowadays, in order tomeet different research purpose, it had developed a variety of methods to generate iPS cells,and the earliest use of retroviral vector-mediated transcription factor to reprogramme thereceptor cells. Because of integration into the host genome, a large number of differentmethods have produced. In this study, a retroviral vector system, respectively, using themethod of plasmid-mediated systems and cloned pigs L-Myc gene on cell reprogramming,laid the foundation for the lower cell tumorigenicity.1. Induction and identification of somatic cells reprogramming with retroviral vectorsIn this study, we generated Bamei porcine induced pluripotent stem cells (piPS cells)with four pluripotent transcription factors, Oct4, Sox2, Klf4and c-Myc from Bamei porcineembryonic fibroblasts (PEF). These cells had flattened shape, neat edge, high nuclearcytoplasmic ratio, and positive alkaline phosphatase (AP) staining. Karyotype analysisshowed that the induced cells had the same number of chromosomes with the normal cells ofPEF cells. They could be cultured longtime in vitro stability, and the shape of clones remainedunchanged. These cells were positive for OCT4, SOX2, TERT, and SSEA-4genes, butnegative for SSEA-1, TRA-1-60, TRA-1-81. In vitro differentiation experiments, the iPS cellscould form embryoid bodie, and had the ability to differentiate into three mesodermal. In theprocess of induction differentiation, we add inducer VC and RA, the results showed that thedifferentiation would be more significant after add inducer RA.2. Induction of somatic cells reprogramming with plasmid vectors In this study, we construct an Oct4/Sox2co-expression vector (pOct4/Sox2-EGFP)containing EGFP marker, and Oct4/Sox2were controlled by two promoters, respectively. Theplasmid vector pOct4/Sox2-EGFP was successfully constructed by restriction endonucleasedetection. Expression of EGFP marker gene and analysis of real-time PCR data showed thatSox2could express in HEK293FT cells and its expression showed an upward trend.Immunofluorescence assay with Oct4antibody and real-time PCR analysis demonstrated thattransfected cells could express Oct4gene, and the expression had a significant increase intransfected0h to48h. Furthermore, we found that receptor cells expressed NANOG gene byreal-time PCR analysis and immunoblotting, while the untreated receptor cells cannot expressNANOG gene. This result suggested that co-expression of Oct4and Sox2could activateexpression of endogenous NANOG gene in adult cells. Moreover, our study established thefundamental work for the next step research to utilize this plasmid to reprogram somatic cellsinto iPS cells.3. Application in cell reprogramming from porcine L-MycIn this study, a1113bp porcine L-Myc cDNA sequence was obtained by RT-PCR,encoding364amino acids, theoretical molecular weight of40kDa. Bioinformatics analysisshowed that a high degree of homology on L-Myc existing among porcine, human and mouse,and their protein structure and function of domain much the same. These results proved thatthis sequence by molecular cloning was the porcine L-Myc CDs. Constructing fusionexpression vector pEGFP/L-Myc-C1, and the vector-transfected and Western blot showed thatthe cloned porcine L-Myc cDNA could be expressed at the protein level. Then we constructedL-Myc into the retroviral vector, different transcription factors respectively induced porcineembryo fibroblast (PEF) cells. The result showed that the morphology of induced cells varied,and the quantity of positive clones induced with the transcription factors Oct4,Sox2,Klf4,L-Myc(OSKL) was far more than that with Oct4,Sox2,Klf4(OSK). It demonstrates thatporcine L-Myc play a role in cell reprogramming, could be helpful to provide the foundationin the induction of porcine induced pluripotent stem (piPS) cells.