Figure 2

Generation of stable lines with a mutated CTCF binding site. A, The transgene and the location of the mutation E (mutE) are shown. B, Stable lines were generated in K562 human erythroleukemic cells and cultured for more than 100 days, with 19 independent clones carrying the intact promoter (pERb) and 18 independent clones with the core Rb promoter with the mutated CTCF binding site (pERbmutE). The point mutations were defined previously [8]. A representative FACS profile is shown for the active and inactive transgenes, respectively. These are results from two independent sets of transfections. C, A representative Southern blot is shown. For hybridization we used a 741 bp DNA probe corresponding to a portion of the GFP transgene. Genomic DNA from each cell line was digested with SacI restriction enzyme and the minimal hybridization signal corresponds to a band of 4,561 bp. This reference band allows us to determine the integrity of the transgene and the integration of a single- or a multi-copy transgene. MW, corresponds to the molecular marker λ-HindIII.