Figure 2

Screening for well-regulated GFP expression. A. Schematic (not to scale) of the target constructs used. DNA is represented as in Fig. 1. Pale and dark ellipses represent original and improved TRP, respectively. B-I. Flow cytometric profiles (y-axis, counts; x-axis, GFP fluorescence [FLH1]) of clones isolated after transfection with plasmids shown in A. For profiles in B (left only), C, D, F and G the green and yellow traces represent cells grown with or without tet, respectively, and the red traces represent GFP-negative cells (Rht14 or HT1080). B. Left: Clone 6, the most stringently regulated clone isolated after transfection of pTARG1 into HT2 cells. Right: GFP expression profile of Clone 6 at indicated times after addition of tet. C. Four of the most stringently regulated clones isolated after transfection of pTARG2 into HT2 cells. D. Three of the most stringently regulated clones isolated after transfection of pTARG3 into HT2 cells. E. Effect of AZC on GFP expression in T15, a pTARG3-transfected HT2 clone. Cells were cultured with (green) or without (blue) AZC for 24 h, and then for a further 24 h without AZC, before analysis. F. Four of the most stringently regulated clones isolated after transfection of pTARG4 into Rht14 cells. G. GFP expression profile of two heterogeneously expressing clones isolated after transfection of Rht14 with pTARG4. H. Profile of clone Rht14-10 grown continuously with passaging for 14 d or 42 d. I. GFP expression in clone Rht14-19, as measured by immunoblot (top) or flow cytometry (bottom), at the indicated times after addition of tet.