Influence of cell type and silencer topology on silencer activity.

(A) GFP reporter construct for assessment in different cell lines. The reporter plasmid is the same as in Fig 3, except in this case only the constructs using the CMV promoter with and without the T39 silencer were compared. (B) Assessment of reporter expression in different cell lines. Reporter plasmids were linearized and transfected into the indicated cell lines. After 7 days, the level of reporter gene expression was analyzed by measuring the mean fluorescence of the cells expressing GFP by flow cytometry. (C) GFP reporter constructs for assessment of silencer topology. The reporter plasmids are the same as in panel A, except the T39 silencer was inserted either 5' only, or 3' only, of the CMV-GVP cassette. (D) Assessment of reporter expression for assessment of silencer topology. Reporter plasmids were left circular or were linearized and transfected into HepG2 cells. After 7 days, the level of reporter gene mean fluorescence was assessed by flow cytometry as for panel B. Data are shown for the mean ± s.e. from three independent transfections, and are normalized to the mean fluorescence of the construct without the T39 silencer. P values based on one-sided t-test comparing constructs with and without the T39 silencer for each cell line. See S1 Table for details on datasets.