Figure 2

Targeting shRNAs and cDNAs to FRT site in 4T1-1V. (Top) The diagram shows the results of FLP recombinase-dependent insertion of the FRT targeting vector carrying a cDNA and/or siRNA, into the genome. The promoter driving expression of the lacZ-zeo fusion protein before insertion drives expression of the hygromycin resistance gene after insertion allowing hygromycin selection of cells that had undergone targeted insertion of the vector. (Bottom) Cells were cotransfected with the indicated vector and an expression vector encoding FLP recombinase. Cell pools and clones were isolated from the transfected cells and assayed for luciferase expression as described in MATERIALS AND METHODS. Light emission in arbitrary units per milligram of cell protein is shown for pools and clones transfected with empty targeting vector or targeting vector encoding luciferase shRNA. Error bars represent standard deviations for empty vector (n = 6) and luciferase siRNA vector (n = 7) transfected clones.