Establishment Of STO Cell Lines Stably And Efficiently Expressing Green Fluorescent Protein And Mouse Leukemia Inhibitory Factor

Objective: To establish the STO cell lines stably and efficiently expressing green fluorescent protein(GFP)and mouse leukemia inhibitory factor(m LIF),and try to culture the mouse embryonic stem cells(m ESCs)with the established STO-GFP-m LIF cells as the feeder layer.Methods: The lentiviral vector containing GFP and m LIF and anti-puromycin gene were validated by sequencing,which was used to transfect the HEK293 F cells with lentiviral packaging vectors ps PAX2 and VSV-G to generate the lentiviral particles that was transduced into STO cell line.The cell lines stably and efficiently expressing GFP and m LIF genes were screened by puromycin.The expression level of the inserted exogenous m LIF gene was tested by Western blot and ELISA.STO-GFP-m LIF cells were treated with different concentrations(5,10,15,20?g/ml)of mitomycin C for different time periods(1.5,2.5,3,3.5 hours)to prepare feeder layers.Cell proliferation was detected by MTT method in all groups.Then m ESCs were cultured on mitomycin C treated feeder layer and the growth of cell colonies was observed by microscope.Results: Fluorescence microscopy can detect GFP expression in lentivirus-infected HEK293 F cells.Western blot and ELISA results revealed the expression level of the inserted exogenous LIF gene of the STO-GFP-m LIF cell lines was higher than that of the STO(P < 0.01).Treatment with 10 ?g/ml mitomycin C for 3 hours showed optimal effects on inhibiting the proliferation of STO-GFP-m LIF cells,with which m ESCs can develop into typical“bird nest”shaped colony of stem cells.Conclusion: The STO-GFP-m LIF cell line stably and highly expressing green fluorescent protein and mouse leukemia inhibitory factor gene was obtained after the lentiviral particles was prepared by lentiviral vector carrying GFP-m LIF-puro gene and lentiviral packaging vector and then transduced into the STO cells,which also can maintain the undifferentiated state of m ESCs as the feeder cells.