Background & objective: Analysis of gene transfer and expression is conventionally inferred from the percentage of positive cells expressing reporter gene in total cells, referred as transfection rate, by investigators counting under a microscope or fluoroscope, which was called as manual counting. But in many cases, it is not accurate and easily influenced by the subjectivity of observer. This study was designed to seek a convenient method to assess objectively and accurately the efficacy of gene transfer and expression.
Methods: Hepatocellular carcinoma(HCC) HepG2 cells were infected with a recombinant adenovirus expressing green fluorescent protein(AdCMV/GFP) at a series of multiplicities of infection(MOIs). 24 h later, the transfection rates were assessed by manual counting under fluorescent microscope. Meanwhile, besides transfection rates, fluorescent indices(FIs) which indicated the efficiency of gene transfer and expression were analyzed by flow cytometry (FCM). Transfection efficiencies of AdCMV/GFP to HCC Hep3B, Bel7402, SMMC7721 cells and nasopharyngeal carcinoma CNE-2 cells were also tested by FCM.
Results: Although transfection rates by FCM were slightly higher than that by manual counting, both were logarithmic correlative with vector doses. The stirring was that FIs by FCM showed compellent linear correlation with vector doses (r = 0.9984, P < 0.001). The efficiency of gene transfer in other cells by FCM were similar to that in HepG2.
Conclusion: The efficiency of gene transfer and expression in mammalian cells can be easily analyzed by flow cytometry, which is more sensitive, objective, and accurate than manual counting, especially in assessing the efficiency of multiple gene transfer (multi-copies per cell) and expression.