PMC

Schematic of the Cre-mediated recombination to create the replaced IgH at the endogenous locus. The pentagon represents the addition of Cre, and the arrows demonstrate the transfection of WT A to create the primary replacement and the transfection of WT B to create the secondary replacement. The asterisk indicates the C-to-G mutation within VDJ of WT B.

Distribution of mutations in WT and HS clones. Distribution of total mutations in the 458-bp VDJ region of HS A and B clones compared to WT A. The red line indicates the boundary of the hot spot cluster. Some sequences were dominated by a single highly mutated site which was removed from the analysis and replaced by the blue line in order to display the results on the same y axis scale. The black boxes represent the WGCW motifs. In order to increase the number of mutations examined, additional sequences from WT A and HS A and B subclones not shown in the previous figures and were added to this figure.

Insertion of a cluster of hot spots into the endogenous heavy chain gene of Ramos cells increases V region mutation. (A) Schematic of the replaced IgH locus with primer pairs used for amplification and sequencing indicated by arrows (P3 to P8). (B) Relative AID transcript levels in the various subclones shown in the bar graph with standard deviations. (C) A bar graph showing the frequencies of mutations depicted in Table 1 from the promoter, V, and constant regions. Error bars represent the standard deviations between 2 clones. (D) Pie charts representing the number of mutations per sequence; the number of sequences analyzed is depicted in the center of each circle.

Schematic of the recombinase-mediated cassette exchange strategy. (A) Homologous recombination to create the Hyg-TK Ramos cell line. A schematic of the productive endogenous IgH allele includes the leader exon (L), the variable region (VDJ), Eμ, Sμ, and Cμ. The 5′ and 3′ homologous arms, the lox sites, and the location of the Hyg-TK gene are depicted in the targeting construct. (B) Southern blot of 5 independently isolated Hyg-TK RMCE clones and a control (lane C) IgM⁺ wild-type non-RMCE Ramos clone. DNA was digested with NotI and NsiI, shown in panel A, and hybridized with a Cμ probe depicted by the black diagonal line in panel A. (C) PCR analysis using genomic DNA of the same clones described in panel B using primers P1 and P2 shown in panel A.