Over-expression Of Sox2 On Reprogramming Of Bovine Fetal Fibroblast Cells

Sox2 is one of SOX(SRY-related high mobility group box,SOX)family members,an important transcription factor for the reprogramming of somatic cells into pluripotent stem cells.In addition to maintaining the spontaneous replication,proliferation and pluripotency of embryonic stem cells,Sox2 also participates in the proliferation and differentiation of various cells such as neural progenitor cells and endoderm.In order to explore the role of Sox2 in the reprogramming process of bovine fetal fibroblast cells,this project was to investigate effects of overexpression of Sox2 gene on the early stage reprogramming of bovine fetal fibroblast cells during cultured in the medium supplemented with specific small molecule compounds.First,primary bovine fetal fibroblast cells were derived and cultured,When the cell lines grew stably with correct karyotype,the cell lines were ready suitable for the experiments.Then,bovine Sox2 gene and Puro gene were cloned by PCR and connected to the pGEFP-N2 vector,and two expression vectors were constructed: pEGFP-N2-Puro and pEGFP-N2-Sox2-T2A-Puro-T2 A.These two expression vectors could express GFP and Puro,or Sox2 as experiemental group,Puro and GFP as the control group.On this basis,the two vectors were introduced into bovine fetal fibroblast cells by electric transfection,and two stable transfected cells were obtained by Puromycin drug screening.The cells transfected with the pEGFP-N2-Puro vector were referred to as GFP cells,and those transfected into the pEGFP-N2-Sox2-T2A-Puro-T2 A vector were referred to as N2 SP cells.Finally,the above bovine fetal fibroblasts were reprogrammed with induction medium respectively,and the cells(0,3,5,7 days after induction)were collected for the following identification: growth curve,cell cycle detection,and karyotype analysis.The two transgenic cell lines were cultured in the medium supplemented with specific small molecules to induce reprogramming of bovine fibroblast cells and cells were collected on different days(0,3,5,7 days and bovine iPS-like cells),respectively.Subsequently,alkaline phosphatase staining,RT-PCR detection,qRT-PCR detection,immunofluorescence staining,western blot were performed with the collected cells.The data including expression of Sox2 gene were collected and observed or analyzed.The results showed that on the early stage reprogramming process of bovine fetal fibroblast cells,overexpression of Sox2 gene could slow down the cell cycle and increase the proportion of G0/G1 phase cells and decrease the proportion of cells in S phase.Loss of chromosomes in all the cells was not found during reprogramming(2N=60).Moreover,overexpression of Sox2 in the cells could significantly increase the proportion of alkaline phosphatase-positive cells.The results collected from the RNA and protein analysis showed the same tendency,namely the expressions of pluripotent genes(Oct4,Sox2 and Rex1)and epithelial cell related genes(Ocln and Cdh1)increased,while overexpressed Sox2 in the cells,whereas Nanog did not.However,the expressions of interstitial cell related genes(Cdh2,Zeb1,Zeb2,Snail and Slug)seemed to down-regulated.The above results show that overexpression of Sox2 could promote the interstitial-epithelial transformation(Mesenchymal-epithelial transition,MET)of bovine fetal fibroblast cells and promote the reprogramming process to some extent.These results may provide some useful information for further investigation on somatic cell reprogramming in farm animals.