Construction of induced mouse pluripotent stem cells by piggyBac transposon and its identification / 吉林大学学报(医学版)

ABSTRACT

Objective:To investigate the effect of piggyBac transpon,as a carrier of four defined transcription factors Oct4,Sox2,Klf4 and c-Myc,in the reprogramming of mouse embryonic fibroblasts (MEFs)to induced pluripotent stem cells (iPSCs).Methods:The MEFs were isolated from Oct4-GFP fetal mice and transfected by piggyBac transposon with four factors (Oct4,Sox2,Klf4 and c-Myc).The morphological changes of clones were traced with microscope during the process of induction.The chromosomes were analyzed to evaluate the karyotypic variation of iPSCs.The mRNA expressions of Oct4, Nanog and FGF4 associated with embryonic stem cells (ESCs)in the iPSCs of mice were tested by RT-PCR;the protein expressions of SSEA-1,Nanog and Alkaline phosphatase in the iPSCs of mice were determined by flow cytometry,immunofluorescence and AP staining.The iPSCs were transplanted into the NOD-SCID mouse groin,4 weeks later,the teratomas were removed for HE staining and the differentiation of tissue was observed.Results:The iPSCs were successfully obtained from MEFs by piggyBac carrying Oct4,Sox2,Klf4,and c-Myc.The round or oval iPSCs clones were similar to ESCs with clear boundry and large dense nuleus.The iPSCs showed the normal karyotypic and expressed the marker genes (Oct4,Nanog and FGF4)and proteins (SSEA-1,Nanog and AP)of ESCs.Teratomas containing three germ layers were formed in NOD-SCID mice after tanspalantation of iPSCs.Conclusion:The iPSCs are reprogrammed from MEFs by piggyBac transposon with four transcription factors-Oct4,Sox2,Klf4 and c-Myc,and the iPSCs with normal karyotype possess the characteristics of ESCs.