Mice were housed in ventilated rack caging in a pathogen-free facility. The Institutional Animal Care and Use Committee of Memorial Sloan-Kettering Cancer Center approved animal use protocols. Mice were maintained on a mixed C57BL/6 and 129Sv background. For mice carrying an inducible Rad50
), genotypes were determined using the primers TGTTCATGATCCCAAGGTAATGGTGTCT (sense), and TCAGAGAACTCATTGTGGAGTCAATTCT (antisense). Rad50−
genotypes were determined using CGCTGTTAAACAGTACTGTCCG (sense) and the antisense primer from the Rad50ind
PCR above. All other genotype strategies were described previously (3
Details of the Rad50ind
targeting construct will be provided upon request. Targeting and Southern blot analyses of embryonic stem cell clones were carried out using previously described methods (37
Ear fibroblast derivation.
Mice were anesthetized with isoflurane, and ear tissue was collected using sterile scissors. Ear fragments were rinsed twice each in 70% ethanol and phosphate-buffered saline (PBS) supplemented with 100 μg/ml kanamycin. Tissue was transferred into 0.3 ml of protease solution (4 mg/ml each of collagenase D and dispase in Dulbecco's modified Eagle's medium [DMEM]; filter sterilized), cut into pieces, and incubated at 37° for 45 min. DMEM (1.5 ml) containing 10% fetal bovine serum (Gemini), 1× glutamine, and 5× antibiotic-antimycotic solution was added, and samples were incubated at 37° overnight. Cells were dissociated by pipetting, passed through a 40-μm-pore-size cell strainer, and plated in DMEM as above except using 1× antibiotic-antimycotic solution. Cells were passaged upon reaching confluence and immortalized via transfection with a plasmid expressing simian virus 40 (SV40) large T-antigen. Transformed cells were grown in DMEM plus 10% cosmic calf serum (CCS; HyClone).
Rad50 deletion in cultured cells.
Adenovirus was commercially obtained (University of Iowa Gene Transfer Vector Core), and cells were infected in suspension at 2.5 × 106 cells/ml at a multiplicity of infection of 50 under low-serum conditions (2% CCS in DMEM) with 5 μg/ml Polybrene for 2 h at 37° on a rotating shaker.
Lentiviral production, concentration, and determination of titers were carried out using established methods (12
). For infections using a lentivirus-Cre-
green fluorescent protein vector, cells were infected in suspension at 1 × 106
cells/ml at a multiplicity of infection of 10 in DMEM plus 10% CCS with 5 μg/ml polybrene. Tubes were spun at 1,900 rpm (~600 × g
) for 90 min, with occasional stops to manually resuspend cells. After viral infection, cells were plated and grown in DMEM containing 10% CCS.
Western blotting was carried out on 40 μg of protein lysates prepared by subjecting cells to three freeze-thaw cycles in NETN buffer (20 mM Tris, pH 8, 150 mM NaCl, 1 mM EDTA, and 0.5% NP-40 plus protease inhibitors). Binding and washing steps were done in 5% milk and PBS-Tween 20 buffer. Rabbit anti-Rad50 polyclonal (1:5,000; custom Petrini lab antibody m84-7) and antiactin mouse monoclonal (1:1,000; AC-40; Sigma) primary antibodies and species-specific secondary antibodies (Pierce) were used, and horseradish peroxidase signal was detected with ECL Plus reagent (Amersham).
For analysis of nuclear aberrations and γ-H2AX foci, cells were seeded onto multiwell slides (Erie Scientific), fixed with 4% paraformaldehyde (PFA), permeabilized (50 mM NaCl, 3 mM MgCl2, 200 mM sucrose, 10 mM HEPES, 0.5% Triton X-100), blocked with 10% fetal bovine serum in PBS, incubated with rabbit anti-γ-H2AX polyclonal antibody (1:300; Upstate) for 2 h, washed, incubated with secondary antibody for 1 h, washed, and stained with DAPI (4′,6′-diamidino-2-phenylindole; Sigma). Slides for this and all subsequent experiments described were scored blind as to the status of genotype. Images were captured on a Zeiss Axiovert microscope using a charge-coupled-device camera (Hammamatsu) and Volocity software (Improvision). For each sample, >100 nuclei were scored.
Metaphases were prepared from cultures treated with colcemid (2 × 10−7 M) for 1 h. Cells were trypsinized, hypotonically swelled (0.075 M KCl) for 7 min at 37°, fixed, washed in ice-cold methanol-acetic acid (3:1), and dropped on slides. Slides were stained with Giemsa (Sigma) for 10 min and rinsed with distilled water, and coverslips were mounted with Permount (Fisher). For each sample, >40 spreads were scored.
For fluorescence-activated cell sorter analysis of the sub-G1 population, cells were harvested, fixed in 70% ethanol, and stained with propidium iodide (Sigma). For each sample, >10,000 events were collected. Data were analyzed using FlowJo software.
Rad50 cDNA-expressing cultures were derived for colony assays via transfection and stable selection of cells bearing an Rad50 expression vector. The assay was done in duplicate using virus-Cre-infected and uninfected control cultures. Plates were stained with crystal violet, and colonies were tabulated after 18 days.
Telomere fluorescence in situ hybridization (FISH) staining was performed on metaphase spreads prepared as outlined above. Slides were then rehydrated in PBS, treated with 0.5 mg/ml RNase A in PBS at 37° for 10 min, rinsed in PBS, dehydrated through a series of ethanol washes (70%, 90%, and 100%), and air dried. A hybridization mixture (10 mM Tris-Cl, pH 7.2, 70% formamide 0.5% block reagent [Dupont NEN], 1:1,000 TelG-Cy3 peptide nucleic acid probe [Applied Biosystems]) was added, coverslips were applied to the slides, and DNA was denatured at 80° for 3 min. Probes were hybridized at room temperature for 2 h in a dark, humid chamber; slides were washed two times for 15 min in wash I (70% formamide, 10 mM Tris-Cl, pH 7.2, 0.1% bovine serum albumin), two times for 5 min in wash II (100 mM Tris-Cl, pH 7.2, 150 mM NaCl, 0.08% Tween, with DAPI added to the second wash), dehydrated in ethanol as above, and dried; coverslips were mounted. A total of >1,000 metaphase chromosomes from a minimum of 15 spreads were analyzed.
For immunofluorescence-FISH, cells were seeded on multiwell slides, fixed for 15 min in 2% PFA, washed in PBS, and blocked for 30 min (1 mg/ml bovine serum albumin, 3% goat serum, 0.1% Triton X-100, 1 mM EDTA). Slides were incubated at room temperature for 3 h with 53BP1 antibody (1:1,000 diluted in blocking solution; Novus), washed, incubated with secondary antibody for 2 h, fixed for 5 min in 2% PFA, and washed in PBS. Slides were dehydrated and denatured for 5 min each under conditions as described for telomere FISH. Slides were hybridized overnight at room temperature using a TelC-fluorescein isothiocyanate probe (1:1,000; Applied Biosystems). Slides were washed two times for 15 min in FISH wash (70% formamide, 10 mM Tris-Cl, pH 7.2) and three times in PBS, with DAPI added to the second wash. For IR-treated samples >1,000 53BP1 foci from at least 35 cells were analyzed for colocalization with telomeres; for Cre-treated Trf2ind/ind (Trf2-deficient) and Rad50Δ/ind (Rad50-deficient) samples, >200 foci from at least 18 cells were analyzed; for untreated samples and Rad50+/ind (Rad50-proficient) Cre-treated samples, 53BP1 focus-positive cells were rare, but >65 foci from at least 11 cells were analyzed.
Partial hepatectomy (PH) and liver regeneration analyses.
Mx-Cre-mediated Rad50 deletion was induced by injecting 6- to 12-week-old mice twice with 400 mg of poly(I)·poly(C) ([pI-pC] Sigma) intraperitoneally at 48-h intervals. PHs were performed after a wait of >28 days for bone marrow recovery. Mice were anesthetized with isoflurane, an incision was made above the abdomen, the large left and two median lobes of the liver were extruded, and lobes were ligated with silk suture and excised. Incisions were cleaned and stitched, and analgesic was administered. Studies conducted on mice at 2, 3, 4, and 8 days post-PH were performed in triplicate; studies at days 1 and 6 post-PH were performed in duplicate. For proliferative indices, bromodeoxyuridine (BrdU) injections were administered at 24-h intervals (50 mg/g of body weight) up to 3 days post-PH. Livers were used for calculation of regeneration kinetics, genotyping, and histological preparations.
Histological sample preparation and staining.
Tissue samples for histological analyses were fixed overnight at 4°C in 4% PFA, stored at 4°C in 70% ethanol, and then processed for paraffin embedding. Sections (8 μm) were prepared, and slides were processed and stained at the Memorial Sloan-Kettering molecular cytogenetics core for γ-H2AX, phospho-H3, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and Rad50. p16 and p19 staining was carried out by the Memorial Sloan-Kettering pathology core. For γ-H2AX and TUNEL analyses, >200 cells per sample were scored. For phospho-H3 analyses >250 cells per sample were scored for determining mitotic indices, and >45 cells per sample were scored for metaphase distributions. Phospo-H3-positive nuclei with chromatin beginning to condense were classified as prophase; those with condensed unaligned chromosomes were scored as prometaphase; aligned, unsegregated chromosomes were scored as metaphase; condensed chromosomes undergoing segregation were scored as anaphase; and decondensed sister nuclei were scored as telophase.
To assess gait abnormalities in Pcp2-Cre
experiments, mice were conditioned to walk down a corridor, and gaits were recorded by dipping front paws in nontoxic red paint and hind paws in blue paint. Measurements from three to four runs per mouse were averaged. At 4 months, two controls and five mutants were analyzed; at 16 months three controls and four mutants were analyzed. To assess balance, studies were conducted as described previously with two controls and four mutant mice at 16 months (22