A TetO-inducible System For Reprogramming Of Porcine Somatic Cells

pluripotent stem cells was obtained by a retrovirus transcription system carrying fourfactors (Oct4, Sox2, Klf4, c-Myc), and was named as induced pluripotent stem cells (iPSCs).iPSCs have ES (embryonic stem) cells identical characteristics, and is reprogramming ofsomatic cells which is not refered to the ethical issues related to the embryo. In recent years,iPSCs technology is developing rapidly, and becoming a hot research subject in life scienceresearch and clinical applications.Many researchers successfully induced pig somatic cells to iPS cells. Due to the methodsand culture conditions for different laboratories to induce porcine iPSCs are not the same,these iPSCs are different in genetic characteristics. Reprogramming process requires a hightiter of the stoichiometry or transcription factor expression, so that four factors are randomintegrating in the gene which results in iPSCs containing different copies of four factors. Therandom insertion of viral vectors makes it difficult to cell reprogramming mechanismsresearch and alternative studies using small molecule factors. The lentivirusTetO-FUW-OSKM and FUW-M2rtTA simultaneously infected porcine Embryonic Fibroblasts(PEF).The primitive iPSCs were induced in iPSCs culture medium with Dox (deoxycycline).Subsequently, the primitive iPSCs differentiated to fibroblast-like cells, called TetO-PEF. TheTetO-PEF containing TetO-FUW-OSKM and FUW-M2rtTA will be direct reprogramming toiPSCs without further infecting. The porcine somatic cells were reprogrammed by TetOinducible system. The copy number of four factors integrated in TetO-PEF cell lines is thesame, which is solve the problem of varying copy number of four factors. Exogenous factorsare activated by adding Dox to start cell reprogramming. It is a new experimental platform forthe study of the mechanism of reprogramming pig, pig iPSCs optimization of cultureconditions, small molecule compounds to induce reprogramming research.The main conclusions were obtained in this study:1. Establish induced reprogramming TetO primitive cell linesThe experiment obtained porcine embryonic fibroblasts. The TetO inducible expressionsystem plasmid TetO-FUW-OSKM and FUW-M2rtTA packaged into viral infects PEF cells,and it will be reprogrammed to iPSCs in the iPSCs medium with Dox. The primitive iPSCs were AP staining positive, and could passage.without serum. After PCR, RT-PCR testing,confirmed the successful integration of exogenous gene and expression of exogenous fourfactors.2. Establish a TetO induction system for secondary fibroblast direct reprogramming toiPSCsIn this study, the TetO-PEF which containing TetO-FUW-OSKM and FUW-M2rtTA wasobtained from the dedifferentiation of EB (Embryoid Body, EB) and the EB was from theprimitive iPSCs. Since the primitive iPSCs has consistent genotype, so does the TetO-PEF.The TetO-PEF could be fast reprogrammed to the secondary iPSCs with Dox.Inimmunofluorescence staining, the secondary iPSCs showed high expression of OCT4,SOX2, SSEA-1, TRA-1-60, weak expression of SSEA-4, but no expression of TRA-1-81.