Seckel MEFs show rapid reduction in cell proliferation and the onset of senescence by the second passage after isolation (Murga et al., 2009). Because differences in apoptosis, senescence, and proliferation rates may affect telomere length measurements, we were careful to always use only cells in the first passage after har- vest. Moreover, by restricting our analysis to metaphase cells de- rived by mitotic arrest of actively dividing MEF cultures, we exclude apoptotic and senescent cells from analysis. In conclusion, we show in this study that severe reduction in ATR levels does not impair telomerase recruitment to short telo- meres but increases telomere fragility and telomere recombina- tion, indicating that the important DDR checkpoint kinases ATM and ATR function differently in telomere maintenance in mam- mals and yeast. Given the important role of ATR in preventing telomere fragility and the occurrence of chromosomes with mul- tiple telomere signals, our results suggest that telomere dysfunc- tion associated with ATR deficiency may in part be responsible for some of the pathologies associated with Seckel syndrome. Materials and methods Generation, genotyping, and handling of mice To generate ATRS/S/Terc/ mice, ATRS/S mice (Murga et al., 2009) were crossed with Terc/ animals (Blasco et al., 1997), and resulting double heterozygous breeding pairs were used to generate G1 of single Terc/ and double ATR+/S/Terc/ animals. G2 mouse colonies were generated by intercrossing G1 ATRS/+/Terc/ pairs. To generate ATR+/+/G3 Terc+/*, ATR+/+/G3 Terc/*, ATRS/S/G3 Terc+/*, and ATRS/S/G3 Terc/* mice, we crossed ATR+/S/G2 Terc/ males with ATR+/S/Terc+/ females. All mice analyzed were from a mixed 129Sv/C57BL6 background. Terc and Seckel mice were genotyped as described previously (Blasco et al., 1997; Murga et al., 2009). Mice were maintained at the Spanish National Cancer Cen- tre (CNIO) under specific pathogen-free conditions in accordance with the recommendations of the Federation of European Laboratory Animal Sci- ence Associations. CT analysis of 10-wk-old mice was performed in the Molecular Imaging Unit at the CNIO using a CT scanner (VISTA; GE Healthcare). Bone density was determined for both proximal and distal fe- murs and expressed as Hounsfield units. Isolation of MEFs MEFs were isolated from day 13.5 embryos as described previously (Blasco et al., 1997), maintained in DME supplemented with 10% (vol/vol) fetal bovine serum and antibiotics/antimycotics, and grown in a 5% CO2 humidified incubator at 37°C. All MEF experiments used cells from the first passage after isolation. Human Seckel cell lines ATR wild-type and ATR Seckel human cell lines were obtained from the Coriell Cell Repositories. Western blotting Whole cell extracts were prepared from primary MEFs, and Western blots were performed as described previously (Martínez et al., 2009a). After transfer, membranes were incubated with an anti-ATR antibody (1:500; AbD Serotec) and anti–-actin monoclonal (1:10,000; Sigma-Aldrich). Anti- body binding was detected using enhanced chemiluminescence after incu- bation with horseradish peroxidase–coupled secondary antibody. Telomerase repeat amplification protocol (TRAP) TRAP was performed as described previously (Siegl-Cachedenier et al., 2007). In brief, primary MEFs were trypsinized and washed in PBS, and S-100 extracts were prepared in lysis buffer with 10% CHAPS. Protein concentration was determined by the Bio-Rad Laboratories protein assay, and two protein concentrations were used for each sample (5 and 1 µg). Extension and amplification by PCR reactions and electrophoresis were performed as described previously (Blasco et al., 1997). A negative control was included by preincubating each MEF extract with RNase for 10 min at 30°C before the extension reaction. An internal control for PCR efficiency was included. Telomere length analysis by Q-FISH Q-FISH was performed on metaphases harvested by incubating MEFs or ATR-deficient human cells with 0.1 µg/ml colcemid (Invitrogen) for 4 h at 37°C followed by fixation in methanol/acetic acid (3:1). Q-FISH on chromo- some metaphase spreads was performed as described previously (Herrera et al., 1999; Samper et al., 2000); metaphase spreads were stained with Vectashield mounting medium for fluorescence with DAPI (Vector Laborato- ries). Telomere fluorescence values were extrapolated from the telomere fluorescence of lymphoma cell lines LY-R (R cells) and LY-S (S cells) with known telomere lengths of 79.9 kb and 9.7 kb, respectively. Images were recorded at room temperature using a camera (CCK; Cohu, Inc.) on a fluores- cence microscope (DMRB; Leica) with a mercury vapor lamp source (CS 100 W-2; Philips) with a 100× oil objective to get a 1,000 magnification. They were captured using Q-FISH software (Leica) in linear acquisition mode. TFL-Telo software (a gift from P. Lansdorp, BC Cancer Agency, Van- couver, British Columbia, Canada) was used to quantify fluorescence intensity of telomeres (Zijlmans et al., 1997). Images from littermate ATR+/+/G3 Terc+/*, ATR+/+/G3 Terc/*, ATRS/S/G3 Terc+/*, and ATRS/S/G3 Terc/* metaphases as well as from human ATR wild-type and ATR Seckel cells were captured on the same day, in parallel, and blindly. Combined Q-FISH and SKY After Q-FISH image capture, coverslips were removed, and slides were washed in PBS for 10 min. Painting probes for each mouse chromosome were generated, labeled, hybridized, and washed according to the man- ufacturer’s protocol (Applied Spectral Imaging) as described previously (García-Cao et al., 2004). The same metaphases previously captured for Q-FISH were relocated, and SKY images captured and processed ac- cording to Samper et al. (2001b). Images were recorded at room tem- perature with a Spectra Cube (SD300; Applied Spectral Imaging) mounted on an imaging microscope (Axioplan 2; Carl Zeiss, Inc.) with a 100× oil Iris objective to get a 1,000 magnification. They were captured using the SkyView 1.6 software (Applied Spectral Imaging) and pro- cessed with the Case Data Manager 5.5 software (Applied Spectral Imaging). Y chromosomes were identified by reverse DAPI banding in the original Q-FISH images. Cytogenetic analyses At least 56 metaphases per genotype were analyzed for chromosomal ab- errations by superimposing the Q-FISH telomere image on the DAPI image using TFL-Telo. Analysis was performed blindly. Metaphases were scored for RLFs (p:p arm fusions), dicentrics (q:q arm fusions), p:q arm fusions, rings (chromosomes with two fused chromatids without telomere signal at the fusion point), chromatid fusions (fusions between chromosomes by one chromatid), complex aberrations, chromatid breaks (gaps in chromatids with an identifiable corresponding chromosome), and fragments (with two telomere signals or less without an identifiable corresponding chromo- some). For MTS analysis, primary MEFs and human ATR wild-type and ATR Seckel cells were treated with aphidicolin at 0.5 µM for 24 h when indi- cated, as described previously (Martínez et al., 2009b). CO-FISH MEFs were grown at 37°C in DME containing 10 µM 5-bromo-2-deoxy- uridine (Sigma-Aldrich) and allowed to replicate their DNA once. 1 µg/ml colcemid was added for the last 4 h. Cells were recovered, and meta- phases were prepared as described previously (Samper et al., 2000). CO- FISH was performed as described previously (Bailey et al., 2004; Gonzalo et al., 2006) using a Cy3-labeled (TTAGGG)7 probe followed by a Rhoda- mine green–labeled (CCCTAA)7 probe (Applied Biosystems). Images of metaphase spreads were captured at room temperature using a CCK cam- era on a DMRB fluorescence microscope with a mercury vapor lamp source (CS 100 W-2) with a 100× oil objective to get a 1,000 magnification. They were recorded using the Q-FISH software. Global SCE determination MEFs were grown at 37°C in DME containing 3 µg/µl 5-bromo-2- deoxyuridine for two rounds of replication. Colcemid was added at a concen- tration of 1 µg/ml for the last 4 h of incubation. Cells were then recovered, and metaphase chromosome spreads were prepared as described for the Q-FISH (Samper et al., 2000), and genomic SCEs were visualized using an adapted fluorescence plus Giemsa protocol as described previously (Jaco et al., 2008). Metaphases on slides were immersed in 5 µg/ml Hoechst 333258 solution (Invitrogen) and washed with abundant water. The slides were exposed to UV light for 15 min in the presence of 2× SSC and washed with water. Then, they were stained with Giemsa solution (Merck) for 2 min Telomere defects in ATR-deficient mice • McNees et al. 649