Generation of mutations in BRCA2 in a BAC clone
BAC RP11-777I19, containing full-length human BRCA2
was used to generate mutations. The mini-lambda-based recombineering system was introduced into BAC containing DH10B cells as described previously26
. The loxP
site was deleted and loxP
511 was replaced with a neomycin resistance gene under the control of Pgk
promoter to enable a positive selection of transgenic ES-cells. Mutations were introduced using the oligonucleotide-based “hit and fix” BAC recombineering method as described previously27
. All mutations were confirmed by sequencing.
Generation of Brca2-null ES-cells carrying a mutant transgene
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 EcoR
I 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 EcoR
I fragment (). 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
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 EcoR
V fragment corresponding to the mutant allele (ko) and a 4.8 kb fragment corresponding to the wild-type or conditional allele (cko) ( and Supplementary Figure 3
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.
For Western blot analysis proteins were extracted in RIPA buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 0.25% sodium deoxycholate, 1 mM sodium fluoride, 1 mM orthovanadate). NE-PER buffer system (Pierce) was used to isolate nuclear and cytoplasmic protein fractions. Proteins were then separated on a precast NuPAGE 4–12% Bis-Tris gel (Invitrogen) in MOPS SDS running buffer (Invitrogen). The following antibodies have been used: BRCA2 (Ab-2, Calbiochem, 1:500), actin (Ab-5, NeoMarkers, 1:1,000), p53 (CM5p, Novocastra), p21 (F-5, Santa Cruz). Secondary antibodies were goat anti-rabbit IgG-HRP (sc-2004, Santa Cruz) at 1:5,000 dilution and goat anti-mouse IgG-HRP (sc-2005, Santa Cruz) at 1:2,000 dilution. ECL Plus Western Blotting Detection system (Amersham) was used for chemiluminescent detection.
Genotoxin sensitivity assay
8,000 ES-cells per well (16,000 cells per well for Y3308X and E3309X mutants to compensate for lower seeding and growth efficiency) were seeded in 96-well plates in 2–4 replicas. Drug treatment started 18 hours later (concentrations indicated in Supplementary Figure 5
online). For UV sensitivity testing 32,000 ES-cells (64,000 cells for Y3308X and E3309X) per well were seeded in triplicates in 24-well plates. Next day cells (without media) were irradiated at indicated doses in a UV-crosslinker (Stratagene) and then cultured in fresh media. For γ-irradiation plates were exposed to a 137
Cs source at 146.3 rad/minute without media change. 72 hours later the relative number of living cells was measured using XTT assay28
. Values were adjusted for “no cells” background.
RAD51 foci formation assay
40,000 cells per well were plated in gelatinized SonicSeal plastic slides (NUNC). 48 hours later slides were irradiated with 10 Gy. Six hours later cells were fixed with 4% paraformaldehyde for 5 minutes, washed twice with PBS and permeabilized in PBS-buffered 0.1% Triton X-100 for 10 minutes. After two additional washes with PBS, cells were blocked in a blocking solution (1% BSA, 0.05% Triton X100, 10% donkey serum in PBS). The antibody staining and imaging was performed as described previously29
Homologous recombination assay
Exponentially growing ES-cells were electroporated with each of the targeting vectors described in Supplementary Figure 6
online. Clones were selected for resistance to hygromycin or puromycin. 96 colonies for each cell line were analyzed for homologous recombination by Southern blot analysis as shown in Supplementary Figure 6
ES-cells were treated with colcemid (Invitrogen) for 1.5 hours to arrest at metaphase. Cells were trypsinized, washed and resuspended in hypotonic solution at 37°C (0.075M KCL) for 15 minutes and fixed in a methanol/acetic acid mixture (3:1 v/v). Air-dried preparations were stained in Giemsa solution (10% Sorensen’s buffer and 2% Giemsa, J.T. Baker). Two hundred well-spread metaphases containing at least 40 chromosomes were examined blindly from each genotype for structural aberrations.
Crystal structure modeling
Crystal structure 1MIU for a C-terminal portion of mouse BRCA2 was retrieved from the protein data bank at http://www.rcsb.org/
and analyzed using Sirius software available at http://sirius.sdsc.edu
and DS ViewerPro from Accelrys Software Inc.
Results obtained from testing mutant ES-cells for sensitivity to genotoxins were further evaluated for statistical significance using ANOVA test. Because Y3308X and E3309X cells showed identical phenotype, they were later tested as one group (n=6) against the group of controls (two WT clones and K3326X polymorphism, n=9) using two-tailed t-test with unequal variance.