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. 2011 Jul 14;7(7):e1002148. doi: 10.1371/journal.pgen.1002148

Figure 1. Gene disruption of the SFR1, SWS1, and BRCA2 loci.

Figure 1

(A) Schematic representation of the endogenous SFR1 locus and gene-disruption constructs carrying the puro or bsr selection marker gene. The solid boxes represent exons, and numbers right above boxes represent exon numbers. Relevant BamHI and EcoRI sites are indicated. (B) Southern-blot analysis of genomic DNA digested by both BamHI and EcoRI was performed using the probe DNA shown in (A). Positions of hybridizing fragments of wild-type (WT) and targeted loci are indicated. (C) Schematic representation of the endogenous SWS1 locus and gene-disruption constructs carrying the puro or his selection marker gene. Relevant EcoRV and NotI sites are indicated. (D) Southern-blot analysis of genomic DNA digested by EcoRV and NotI was performed with the probe DNA shown in (C). (E) Experimental methods to generate BRCA2−/− and RAD51mediator−/−/BRCA2−/− cells. We generated BRCA2−/− cells from conditional mutant BRCA2−/con1 cells. In the minus allele of the BRCA2−/con1 cells, the whole coding sequence is deleted (hereafter called the coding sequence deletion allele). The structures of the conditional-null allele-1 (con1) is shown in (F). Treatment of BRCA2−/con1 cells with 4-OH tamoxifen (TAM) led to the generation of BRCA2−/− cells. To generate RAD51mediator−/−/BRCA2−/− cells, we disrupted one of the RAD51mediator genes in BRCA2−/con1 cells. Exposure of the resulting RAD51mediator−/−/BRCA2−/con1 cells to TAM led to the generation of RAD51mediator−/−/BRCA2−/− cells. (F) Schematic representation of BRCA2 conditional-null allele and the brca2-null allele wherein the whole coding sequences are deleted. The conditional-null allele-1 (con1) shown on top was described previously [33]; the structure of the coding sequence deletion allele is shown in the second row. Treatment of the BRCA2−/con1 cells with TAM causes deletion of the promoter and initiation codon. The relevant EcoRI sites in the conditional-null allele-1, the relevant XbaI sites in the coding sequence deletion allele, and the position of the probes used in the Southern-blot analysis (G) are indicated. The solid boxes and arrowheads represent the exons and loxP signals, respectively. (G) Southern-blot analysis of the conditional allele (left) and the other coding sequence deletion (−) allele (right) in BRCA2−/con1 cells with (+) or without (−) TAM treatment. Southern-blot analysis of EcoRI or XbaI-digested genomic DNA was performed with the probe DNA shown in (F). (H) Western-blot analysis to verify the loss of BRCA2 protein in BRCA2−/− cells derived from BRCA2−/con1 cells.