Figure 4.

BRCA2 ^Δ5+7 expression and functional analysis in mouse ES cells. (a) Sequence of the region containing exons 5 and 7 and the flanking intronic regions deleted from BRCA2. (b) RT-PCR analysis of BRCA2 transcripts in mouse ES cells expressing WT and Δ5+7 BRCA2 transgene using primers from exons 2 and 9. (c) Sequence analysis of the 623bp RT-PCR fragment that lacks exons 5 and 7. (d) Southern blot analysis of HAT^r ES cell colonies obtained after Cre-mediated deletion of the conditional of BRCA2. The upper band corresponds to the conditional allele (cko), and the lower band corresponds to the mutant allele (ko). The sizes of the bands are shown on the left. (e) Detection of conditionally expressed BRCA2 by immunoblot. (f) XTT assay of cells expressing WT or Δ5+7 mutant BRCA2 (C4/F4 and D1/D3) following 48 h of treatment with MMC, cisplatin, camptothecin, MMS and γ-irradiation (IR) to examine their sensitivity to these DNA-damaging agents. (g) Efficiency of homologous recombination (HR) measure by using DR-GFP reporter after generation of I-SceI induced double strand break. Graph shows the percentage of GFP positive cells in WT and two Δ5+7 mutant clones (C4/F4 and D1/D3)