Figure 6: Analysis of TRF1 and DNA damage in chimaeric tissues.

(a) Mean TRF1 intensity in cells bearing normal (GFP-negative) or hyper-long telomeres (GFP-positive) in ES cells used for the generation of chimaeric mice, and in brain, intestine and skin from the chimaeric mice at 6 and 12 months. Tissues were subjected to IF with anti-GFP and anti-TRF1 antibodies. Representative graphs of two independent experiments. (b) Per cent of cells positive for γH2AX in brain, intestine and skin tissue from chimaeric mice at 6 and 12 months. Tissues were subjected to IF with anti-GFP and anti-γH2AX antibodies. Representative graphs of two independent experiments. (c) Representative images of brain tissues as described in b. Scale bar, 10 μm. (d) The graph shows the per cent of P53-positive cells in skin from 1-year-old chimaeric mice bearing hyper-long telomeres for both GFP-positive and -negative cells. Underneath, a representative image of intestine stained for GFP and P53 and in 1-year-old chimaeric mice. The yellow arrowhead indicates GFP stain and the red arrow indicates p53 stain. Scale bar, 50 μm. (e) The graph shows the per cent survival of three different cohorts of mice, chimaeric mice with normal telomere length, chimaeric mice bearing cells with hyper-long telomeres and control mice. (f) The graphs show the percentage of spontaneous tumour incidence in the three cohorts of mice described in e. (g) Chemical carcinogenesis experiment. The graph shows the total number of papillomas in chimaeric mice bearing hyper-long telomres, normal length telomeres or in age-matched mice of the 129S1 and C57Bl6 backgrounds. Representative graph of two independent experiments. (h) The graph shows the percentage of area affected by hyperkeratosis in the mice described in g. Representative graph of two independent experiments. (i) Representative micrograph of back skin of mice affected with hyperkeratosis. (j) E&H images on skin affected with hyperkeratosis. The lesion was diagnosed parakeratotic hyperkeratosis, as no signs of inflammation were observed. n=number of independent clones of ES cells or chimaeric or control mice. Scale bar, 50 μm. The s.e.m. was represented in error bars. Student t-test with the Bonferroni correction was used to calculate the P values, except for the graph of e, where a log rank test was used. E&H, eosin and haematoxylin.