Figure 4. Analysis of double Igκ enhancer knock-in DT40 cells.

A) Gene-targeting scheme with the murine iE/MAR and 3kE shown as orange ovals, for a detailed description of all elements see Fig. 1. The restriction sites are marked (A: AvrII, and B: BamHI), and the red line above the locus denotes the Southern blot probe (P). The asterisk in each Southern blot marks the band that originates from the non-rearranged allele. B) Northern Blots were run using 10 µg of total RNA of wild-type (WT) DT40 cells and each of the indicated cell lines, and hybridized sequentially with IGL and GAPDH probes. One representative example is shown. C) Normalized steady-state transcript levels of IGL were determined by Northern blots analysis. The level in wild-type cells is arbitrarily set to one, and an average of at least four data points obtained for each genotype is shown. D) Average percentages of IgM⁺ cells (twelve subclones per indicate clone) at each time point are shown for each genotype. E) Mutation event frequencies in the IGL (filled bars) and IGH (open bars) loci were determined by sequencing of the VJ or VDJ exon from two independent subclones of the ieK DT40 cells after four weeks of continuous culture. The data for wild type (WT) and ΔME6K were obtained from [13]. Asterisks indicate p<0.005 in a Student's t-test.