 Ku86-, DNA-PKcs–, and PARP-1–deficient mice were obtained from different laboratories, we cannot rule out small differences in the genetic background of the three independent mouse colonies studied here. This may be particularly relevant in the case of telomerase deficiency because the survival of telomerase knockout mice is known to be extremely dependent on the genetic background, due in part to initial differences in telomere length (Herrera et al., 1999). Therefore, to avoid any artifacts due to subtle differences in genetic backgrounds, we strictly followed the breeding protocol described above. Following this breeding protocol, each comparison we make is between littermate mice within a giving mouse colony, which by definition have an identical genetic background. Mice were housed in the barrier area at the National Biotechnology Center (CNB; Madrid, Spain), in which pathogen-free procedures are used in all mouse rooms.
First passage MEFs were prepared and analyzed by Q-FISH as described previously (Samper et al., 2000). Telomere fluorescence was analyzed using the TFL-telo software provided by Peter Lansdorp (British Columbia Cancer Center, Vancouver, Canada).
Metaphases (50 each) from MEFs prepared from littermate embryos (one embryo per genotype) were scored for chromosomal aberrations by superimposing the telomere image (Q-FISH) on the chromosome image (DAPI) using the TFL-telo software, as described previously (Espejel et al., 2002a,b). We define telomere associations as chromosomes with four distinct telomere signals but aligned less than one-half chromatid apart, and telomere fusions as chromosomes joined at their telomeres and showing at least two overlapping telomeric signals (Goytisolo et al., 2001). Such end-to-end fusions may be chromosomes fused by their p-arms (Robertsonian-like fusions) or by their q-arms (dicentrics).
Mouse tissues were fixed in 10% buffered formalin and embedded in paraffin. Paraffin sections (5 μm) were stained with Harris hematoxylin/eosin according to standard procedures, examined at RT by a microscope (Vanus AHES 3; Olympus) using an S Plan fluorite 10× objective, and photographed with a digital camera (model DP 10; Olympus). Proliferating and apoptotic cells were identified using mAbs against Ki67 (1:1,000 dilution of clone MIB-1 from DAKO Cytomation) and active caspase-3 (1:25 dilution of clone C92-605 from BD Biosciences), respectively. Cells positive for Ki67 and active caspase-3 were visualized using peroxidase-coupled secondary antibodies and 3,3′-DAB tetrahydrochloride as chromogen; nuclei were counterstained with hematoxylin according to standard procedures (Espejel et al., 2002b). Photomicrographs were obtained at RT with a microscope (DM LB; Leica) equipped with N-Plan 20× or 40× objectives (Leica), a digital camera (model DC 100; Leica), and IM1000 imaging software (Leica). The relative amount of immunopositive cells was estimated by visual inspection of at least five different sections from two different tissue preparations and scoring for immunopositive cells in at least 10 crypts per section. Age-related pathologies detected in moribund animals are detailed in the Online supplemental material section.
To assess statistical significance, the Fisher exact probability test was performed with the software and according to the guidelines at the VassarStats web site (http://faculty.vassar.edu/lowry/VassarStats.html).
Table S1 shows the effects of Ku86, DNA-PKcs, or PARP-1 ablation on the development of senile lesions in successive generations of telomerase-deficient mice.
We thank R. Serrano for mouse care, E. Santos and J. Freire for genotyping, and M. Serrano for critical comments on the manuscript.
G. de Murcia's laboratory is supported by the Association pour la Recherche sur le Cancer and La Ligue contre le Cancer. M.A. Blasco's laboratory is funded by the MCyT (SAF2001-1869, GEN2001-4856-C13-08), CAM (08.1/0054/01), European Union (TELOSENS FIGH-CT-2002-00217, INTACT LSHC-CT-2003-506803, ZINCAGE FOOD-CT-2003-506850, RISC-RAD FI6R-CT-2003-508842), and the Josef Steiner Award 2003. S. Espejel is supported by a fellowship from the European Union.
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