Reintroduction of a conditionally active EKLF protein into EKLF^−/− cell lines.

(A) Map of the retroviral vector, MSCV-EKLF-ER™-IRES-GFP. The proviral version of the retroviral vector is indicated with relevant restriction sites (i), as well as splice donor (SD) and splice acceptor (SA) sites for generation of the subgenomic bicistronic RNA (ii). EKLF-ER™ and GFP are independently translated (iii) from a bicistronic mRNA by virtue of the IRES. R indicates EcoR1; B, BamH1; X, Xba1; Nc, Nco1; Nt, Not1; LTR, long-terminal repeat; and the bar, the position of the EKLF probe used for Southern blotting. The asterisk indicates the position of the G525R amino acid substitution in the LBD of ER^TM that renders it refractory to estrogen but sensitive to tamoxifen. (B) Dot plots from flow cytometric analysis of GFP expression in B1 and A2 cells after infection with MSCV-EKLF-ER^TM-IRES-GFP. Shown are dot plots of forward scatter (FSC) compared with the intensity of GFP. Nontransduced control cells were used to determine the GFP and FSC analysis quadrants shown. In each case, the percentage of cells expressing GFP is indicated in the top right corner. (C) Green fluorescence of a colony of transduced B1 cells after sorting and cloning of GFP⁺ cells.