Generation Of Safe Bovine Induced Pluripotent Stem Cells Using Recombinant Proteins

Stem cells provide seed cells for the research of regenerative medicine,gene therapy,early embryonic development and tissue renovation because of their self-renew and multipotency.However,because of the difficulties of obtaining materials and culturing,the research of stem cells of large animals,especially embryonic stem cells has been faced with so much problems.Recently,researchers reprogrammed differentiated somatic cells to generate special stem cells,which were called induced pluripotent stem cells,abbreviated as iPSCs.Since some of their properties are similar with ESCs’,they can be considered as stem cells to some extent.In addition,there is no any argument of the ethics and morality with iPS cells,they have been the central issue in stem cell area,even regeneration medicine area.At present,although the techniques generating iPS cells have been mature,there are many kinds of shortcomings with them.Using purified transcriptional factors to generate iPS cells can not only eliminate the effect of exotic genes and oncogene to cell genome,but also generate economic and applicable iPS cells easily and quickly,which has been a better method to generate iPS cells.This research applies five proteins-Oct4,Sox2,Klf4,c-Myc and Nanog to transfect cells directly to generate safe bovine iPS cells.1、Expression,purification and identification of five recombinant reprogramming factors with cell penetrating peptideThis research amplified Oct4、Sox2、Klf4、c-Myc and Nanog genes with the coding sequence of HIV TAT at3’terminal by two-step PCR,and then ligated them to the prokaryotic expression vector pET-28a,and constructed expression plasmids-pET-28a-Oct4-HIV-TAT, pET-28a-Sox2-HIV-TAT, pET-28a-c-Myc-HIV-TAT, pET-28a-Klf4-HIV-TAT, pET-28a-Nanog-HIV-TAT.These plasmids were transformed into E.coli BL21(DE3) competent cells and Isopropyl β-D-1-Thiogalactopyranoside (IPTG) was used to induce the expression,and then purified five proteins using His-tag by His-Ni-NTA resin.And proteins are identified by Western Blotting.2、 Construction of POct4-EGFP reporter cell lines of bovine fibroblast cellsTo construct POct4-EGFP specific reporter vector:to use plasmid pEGFP-C1as the bone vector,and replace CMV promoter with bovine Oct4promoter amplified by PCR,which could examine the generation of the sternness.To add CMV-DsRed phase to the bone vector in order to make sure the efficiency of transfection.At the same time,we add the φDC31integrase identification phase-attB site to the vector for the purpose of integrating the exotic reporter genes into the genome by φC31integrase.By transfecting both φC31integrase and the reporter vector,and screening cells by G418,and then culturing and passaging the cells,we got the reporter cell lines of bovine fibroblast cells.And then if we induced these reporter cells as maternal cells to generate iPS cells,the exotic Oct4promoter would switch on the expression of EGFP gene,and then the cells would express green fluorescence which would help us observe and identify the generation of iPS cells.3、 Generation and identification of bovine iPS cellsAfter adding five enough recombinant proteins to the culture medium of the above reporter cells by period,we got cells expressing green fluorescence,which were our target cells.These cells had the similar morphology and characteristic with embryonic stem cells and had been identified to be iPS cells preliminarily,like APS staining examined that they were positive.Our results showed,this research constructed bovine POct4-EGFP reporter cell lines for the first time，and used these cells as the donor cells to generate safe bovine iPS cells using recombinant proteins,which laid the foundation for the following research of early embryonic development and animal cloning.