A method to detect gene transfection efficacy by flow cytometry

Background Objective:Analysis of gene transfer and expression is conventionally inferred fro m the percentage of positive cells expressing reporter gene in total cells,re ferred as transfection rate,by investigators counting under a microscope or fluoroscope,w hich was called as manual counting.B ut in many cases,it is not accurate an d easily influenced by the subjectivity of ob server.This study was designed to se ek a convenient method to assess obje ctively and accurately the efficacy of gene tran sfer and expression.Methods:Hepatocellular carcinoma (HCC)HepG2cells were infected with a recombinant adenovirus expressing green fluorescent pr otein(AdCMV /GFP)at a series of multiplicities of infection(MOIs).24h later,the transfection rates w ere assessed by manual counting under fluorescent microscope.Meanwhile,besides transfection ra tes,fluorescent indices(FIs)which indicated the efficiency of ge ne transfer and expression were analyzed by flow cytometry(FCM).Transfection efficiencies of AdCM V /GFP to HCC Hep3B,Bel7402,SMMC7721cells and nasopharyngeal carcinoma CNE-2cells were also tested by FCM.Results:Although transfection rates by FCM w ere slightly higher than that by manual c ounting,both were logarithmic corr elative with vector doses.The stirring was that FIs by FCM showed compellent linear correlation with vector doses(r=0.9984,P0.001).The efficiency of gene transfer in other cells by FCM were similar to that in HepG2.Conclusion:The efficiency of gene transfer and e xpression in mammalian cells can be easily analyzed by flow cytome try,which is more sensitive,objective,and accurate than manual counting,especially in assessing the efficie ncy of multiple gene transfer(multi-copies per cell)and expression.