Generation and Characterization of Brca2 Mutant ES Cells

Mouse embryonic stem cells were cultured in M15 media (Knock-out DMEM (Gibco) and supplemented with 15% fetal bovine serum (HyClone), GPS (glutamate-penicillin-streptomycin, Gibco) and 0.1 mM β-mercaptoethanol with SNL feeder cells. 25μg BACμ DNA was electroporated into 10⁷ Pl2F7 ES-cells (for details on generation of these cells from the AB2.2 cell line see Supplementary Figures 1–3 online) in 0.9 ml PBS at 230 V, 500 μF. Transgenic colonies were selected with G418 (Gibco) and analyzed for the presence of BRCA2 gene by Southern blot. Genomic DNA was hybridized with a 581 bp fragment corresponding to intron 1 of BRCA2 as a 5 probe (nucleotides 13,869,785–13,870,365 of NT_024524.13) recognizing a 1,835 bp EcoRI fragment and a 356 bp fragment corresponding to exon 27 of BRCA2 as a 3′ probe (nucleotides 13,953,046–13,953,401 of NT_024524.13) recognizing a 4,275 bp EcoRI fragment (Fig. 1b). Double-positive clones were further tested for expression of BRCA2 by RT-PCR and Western blot analysis. BRCA2 expressing clones were electroporated with Pgk-Cre plasmid4 (link) and 10⁵ cells were seeded in a 100 mm dish with SNL-feeder cells. Recombinant colonies were selected in the HAT media (Gibco) for 5 days followed by 2 days in HT media (Gibco). Viable colonies were analyzed by Southern blot to confirm the loss of the conditional Brca2 allele. A 1.5 kb probe specific to Brca2 exon 11 (nucleotides 5208–6710 of NM 009765) has been used to detect a 2.2 kb EcoRV fragment corresponding to the mutant allele (ko) and a 4.8 kb fragment corresponding to the wild-type or conditional allele (cko) (Fig. 1e and Supplementary Figure 3 online). HAT^r colonies were also stained with methylene blue (2% methylene blue w/v in 70% ethanol for 15 minutes followed by washing with 70% ethanol) to facilitate their quantification.