Induction Of Pluripotent Stem Cells From Sheep Embryonic Fibroblast By Mouse Defined Factors

Considering of important potential application in regenerative medicine field,induced pluripotent stem cells in turn become a hot research in recent years.With further research,induced pluripotent stem cells have been significant progress in the induction mechanism,induction methods,induce efficiency,clinical applications,and creating induced pluripotent stem cell lines of different species and other areas.Currently,induced pluripotent stem cell lines of mice,rats,humans,rhesus monkeys,pigs and other species have been successfully established.However,induced pluripotent stem cells of sheep have relatively few successful establishment of reports and inefficient induction.In this study,a retroviral vector delivery system was used to deliver inducing factor which were mouse-derived Oct4,Sox2,Klf4 and c-Myc(4 pluripotency genes)and miR-200c,miR-302(2 microRNA),and imported into sheep embryos Apex fibroblasts,and then obtained sheep induced pluripotent stem cells,and detected pluripotent.This study laid the foundation of improving the method of establishment sheep iPS cell lines and provening of mechanism of inducing sheep iPSCs.The results were as follows:1.Preparations of inducible facotrs and exprimental cells:we endotoxin-free extracted retroviral vector plasmids carried Oct4,Sox2,Klf4,c-Myc,miR-200c and miR-302,and then further increased the concentration and purity by purifying.Theoretical fragment length was detected after double digestion.We prepared mouse embryonic fibroblasts and sheep embryos Apex fibroblasts by trypsin digestion.Then prepared feeder layer by treatment of mitomycin C.Then recoveryed packaging virus cell Plat-E.All cells were successfully separated,culture,cryopreservation and resuscitation.2.Establishment of sheep induced pluripotent stem cell lines:transfected 6 inducible factors by using liposome,collected and concentrated retrovirus packaged by Plat-E,thereby infecting sheep embryos Apex fibroblasts,and then cultured in embryonic stem cell culture medium containing leukemia inhibitory factor and basic fibroblast growth factor.Five days later,colonies similar to embryonic stem cells were detected.After passaging and selection repeatly,we established sheep induced pluripotent cell lines.3.Detecting of sheep induced pluripotent stem cells:we tested induction pluripotent stem cells obtained by alkaline phosphatase activity and Oct4 immunofluorescence staining,and the results were positive.Thereby extracted genomic DNA of induced pluripotent stem cells,and then amplified methylation island of the promoter region of Sox2 after methylation treatment in order to detect methylation status.It was found that Sox2 promoter region of induced pluripotent stem cells had the degree of methylation of 7.04%,comparing with sheep fibroblasts methylation degree of 72.96%.It indicated that significant demethylation occured after reprogramming.In summary,by using of mouse-drived inducible factors,sheep induced pluripotent stem cell lines were constructed successfully.