Preliminary Study On Establishment Of Bovine Fetal Fibroblasts That KRT18 Gene Labeled By Td- Tomato And Using For IPS Induction

Epithelial to mesenchymal transition(EMT)is a process of transition from epithelial-like cells to mesenchymal-like cells,which is an intermediate state change that has been shown to play an imporant role in cancer development,tissue healing,and iPS induction.During the iPS induction,the main process of EMT is the reverse of fibroblast-cell-epithelial-like cells(MET).In this process,the cell morphology of fibroblasts is gradually lost,transformed into a more closely connected epithelial cell morphology,and expresses the specific marker KRT18,KRT8 and KRT19 of simple epithelial cells.As a simple epithelin in intermediate filament proteins,KRT18 is not only found to have an important role in cancer,but also plays a role in the early embryo development and implantation and the occurrence of MET in the iPS induction.Therefore,the study of the effects of this gene in embryonic development and iPS induction is important for understanding its functions.In this study,the pKlrkt which has constructed in the laboratory was used as the basic vector;the 5' homologous arm in KRT18 the promoter region and the 3' homologous arm were obtained from the fetal bovine fibroblast genome by PCR.Two homologous arms were ligated to the MluI and NheI sites of the basic vector by ligation.Then the td-Tomato plasmid and pKlrkta vector were digested by BamHI and NotI,then ligated together,the pTKlrkt targeting vector that containing a td-Tomato sequence was built.The pTKlrkt targeting vector and the CRISPR/Cas9 that contains specific gRNA sequences was co-transfected into fetal bovine fibroblasts at 200 V,5 ms,and electric shock twice by electroporation.After screening by G418,the monoclonal cells were sorted by flow cell sorting and mouth pipetting,and 122 monoclonal cells were finally obtained.Cross-arm PCR confirmed that 3 corrected target cell lines were identified,named BFF-KRT18-Tomato,and the target efficiency is 2.46%.The three target cell lines maintained the growth rate of the original fibroblasts and could be passaged for more than 5 generations in vitro.In order to study the characteristics of KRT18 during iPS induction,the BFFKRT18-Tomato targeted cell line was used for iPS production.The human Oct4,Klf4,Sox2,C-Myc(h-OKSM),human Oct4,Klf4,Sox2,C-Myc,Nanog(h-OKSMN)and human Oct4,Klf4,Sox2,C-Myc,Nanog,Lin28A(h-OKSMNL)were transfected into the targeted cell line by lentivirus transfection.The CTFR culture system(MTeSR E6,Low fatty acid BSA,rhFGF-2,IWR-1)and LCDM culture system(DMEM/F12,Neurobasal,N-2,B27,L-Glutamine,NEAA,KSR,?-mercaptoethanol,bLIF,CHIR-99021,DiM,MiN)were used for culture of the transfected cells.The cells were collected at day 18 for RT-PCR analysis and td-tomato expression were observed under the fluorescence microscope.The results showed that h-OKSMNL could promote the transformation of fibroblast-like epithelial cells,and neither of the two culture systems could support the formation of iPS cell colonies,but CTFR culture system could maintain the growth of the cells.In summary,the td-Tomato gene can efficiently integrate into the KRT18 gene locus of bovine fetal fibroblasts by CRISPR/Cas9.The established cell lines can serve as a marker in MET research,providing a powerful tool for studying the function of KRT18 gene and the iPS induction process.