The Effect Of Using Recombinant Lentivirus Vector With Human SCL Gene To Transfect Cajal-like Cells In Vitro

Purpose: Use recombinant lentiviral vector of human SCL to transfect ICC-like cells cultured in vitro,to assess the transfection efficiency of the recombinant lentiviral vector.Methods: The ICCs-like cells was isolated from the bladder of guinea pig by collagenase digestion,and immunohistochemistry identified by ICC-like cells specific marker c-kit antibody.Use recombinant lentiviral vector to transfect ICC-like cells cultured in vitro. From using the fluorescent microscope to observed the expression of green fluorescent protein(GFP) in the first The first 2 days、3 days, 5 days after transfecting,and calculating transfection efficiency.The total RNA was extracted at the same time,the expression of SCL in the ICCs-like cells was measured by RT-PCR method.Results: When MOI = 10, 72 hours after transfection,the recombinant lentiviral vector get the highest biological activity and safety,the transfection efficiency of the recombinant lentiviral vector of human SCL up to 85% by observing the expression of green fluorescence protein( GFP) under the fluorescent microscope.The total RNA was extracted in the first The first 2 days、3 days, 5 days after transfecting,use RT-PCR method to measure the expression of SCL in the ICCs-like cells.The experimental group expressed highly specific bands by agarose gel electrophoresis and consistent with the length of the SCL gene,but the normal reference group and positive reference group does not appear specific bands.It is confirmed that the the lentivirus-mediated SCL can be effectively expressed in ICCs-like cells.Conclusion:The recombinant lentiviral vector containing human SCL gene has a high transfection efficiency and can successfully mediated expression of SCL in ICCs-like cells.