Figure 1.
(A) Genomic organization and disruption strategy for Xpg depicting the wild type allele (+), the targeting construct, the targeted allele (fn), the conditional allele after Flp-mediated recombination of Frt sites (f) and the targeted Xpg allele following subsequent Cre-mediated recombination of LoxP sites (−). Exons 2–5 are indicated by black boxes. PCR primers are shown as arrows. (B) Southern blot and PCR analysis of an ES clone showing the correct insertion of the targeting construct. ES cell genomic DNA was digested with EcoRI for Southern blot analysis and hybridized with a 0.9 kb DpnI probe. The wild type (wt) allele yields a 7.4-kb fragment whereas the targeted (tg) allele yields a 4.1-kb fragment. The NheI-digested PCR product shows the 2.3-kb and 2.2-kb bands corresponding with the wt and tg allele, respectively (see also panel A). (C) PCR detection of mouse genotypes using the primers F1, NeoF and R1 as indicated as in A. (D) Immunoblot analysis of extracts from Xpg−/− and wt MDFs using a rabbit polyclonal antibody raised against a peptide conserved between human and mouse XPG. Tubulin is used as loading control. (E) Primary Xpg−/− and wt MDFs, cultured at low (3%) O2 levels were irradiated with the indicated doses of UV-C (left) or treated with the indicated doses of Illudin S for 1 h (right). After 48 h recovery, survival was assessed by cell count. (F) UV-induced UDS in primary Xpg−/− and wt MDFs reveals a severe GG-NER defect in Xpg−/− cells. MDFs were irradiated with 16 J/m2 of UV-C. UDS levels are expressed relative to the non-irradiated wt cells. (G) UV-induced RRS in primary Xpg−/− and wt MDFs reveals a severe TC-NER defect in Xpg−/− cells. MDFs were irradiated with 16 J/m2 of UV-C. 16 h after UV irradiation the wt cells show recovery of RNA synthesis, while Xpg−/− MDFs only show residual activity in nucleoli (rRNA transcription). Arrowheads indicate nuclei. Error bars indicate standard error of the mean. **p<0.01.
Figure 2.
Progeroid characteristics of Xpg−/− mice.
(A) Survival of Xpg−/− mice in a C57Bl6 (red), FVB/N (green) or C57Bl6/FVB F1 hybrid (blue) background; n = 5 (C57Bl6), n = 10 (FVB/N), n = 14 (C57Bl6/FVB F1 hybrid). (B) Average body weight of embryonic 17.5-day old F1 hybrid Xpg−/− and wild type (wt) littermates; n≥12 animals/group. (C) Average body weight of F1 hybrid wt males (black triangles), wt females (black circles), Xpg−/− males (grey triangles), and Xpg−/− females (grey circles); n≥4 animals/group. (D) Left: Photograph of a 7-day old F1 hybrid Xpg−/− and wt littermate, showing no apparent differences except a slightly smaller size. Top right: Photograph of a 14-week old Xpg−/− mouse. Bottom right: Side by side comparison of the same 14-week old Xpg−/− and wt littermate showing a pronounced growth deficiency of the Xpg−/− mouse. (E) Onset of hind limb clasping (orange), tremor (red) and kyphosis (green) with age and survival of F1 hybrid Xpg−/− mice; n = 33 (clasping, tremor and kyphosis), n = 14 (survival). (F) CT-scan of a 16-week old F1 hybrid wt (left) and Xpg−/− (right) mouse showing prominent curvature of the spine (kyphosis) in the Xpg−/− mouse. (G) Bone strength of F1 hybrid Xpg−/− and wt mice analyzed by a 3-point-bending assay of the femur at an average age of 15 weeks; n≥6 animals/group. (H) Cortical (left) and trabecular (right) thickness of the femora of F1 hybrid Xpg−/− and wt mice at different ages; n = 4 animals/group. Error bars indicate standard error of the mean. *p<0.05, **p<0.01.
Table 1.
Xpg−/− mice are born below Mendelian ratio in a C57BL6 background.
Figure 3.
Intestine and liver phenotype of Xpg−/− mice.
(A) Representative images of HE and Ki67 stained small intestine (SI) of 14-week old Xpg−/− and wild type (wt) mice showing no gross morphological differences. (B) Average nucleus size of hepatocytes in the liver of 4- and 14-week old Xpg−/− and wt mice; n≥3 animals/group. Bottom right: magnification of a nuclear inclusion found sporadically in liver sections of 14-week old Xpg−/− mice. (C) Relative mRNA expression levels of several antioxidant genes and the DNA damage response gene p21 in liver tissue of 7- and 14-week old Xpg−/− and wt mice. All values are corrected for TubG2, Hprt, and Rps9 (Table S1) expression as internal standard and normalized to the 7-week old wt expression levels; n = 4 animals/group. (D) Relative expression levels of the somatotrophic genes Ghr, Igf1r, Igf1, and Igfbp3 in liver tissue of 7- and 14-week old Xpg−/− and wt mice. All values are corrected for TubG2, Hprt, and Rps9 expression and normalized to the 7-week old wt expression levels; n = 4 animals/group. (E) Average basal blood glucose levels in groups of 4–7 and 12–18 week old Xpg−/− and wt mice; n≥15 animals/group. Scale bars: 50 µm (A), 10 µm (B). Error bars indicate standard error of the mean. *p<0.05, **p<0.01.
Figure 4.
Increased cell death, degeneration and stress responses in post-mitotic tissues of Xpg−/− mice.
(A) Representative images of GFAP immunostained sagittal neocortex sections of 4- and 14-week old Xpg−/− and wild type (wt) mice showing progressive astrocytosis in Xpg−/− mice. cc: corpus callosum. (B) Quantification of p53-positive cells per mm2 in neocortex (NCx) and cerebellum (Cb) sections of 4- and 14-week old Xpg−/− and wt mice; n = 3 (14 weeks) and the average of five sections of a 4-week old Xpg−/− and wt animal. (C) Representative images of calbindin immunostained sagittal cerebellum sections of 4- and 14-week old Xpg−/− and wt mice. Right panel: Magnification of the areas marked with dotted black boxes. Arrows indicate cerebellar torpedoes. ml: molecular layer, gl: granular layer. (D) Quantification of TUNEL-positive cells per cm2 in neocortex and cerebellum sections of 4- and 14-week old Xpg−/− and wt mice; n≥3 animals/group. Arrows indicate positive cells. (E) Relative mRNA expression levels of the antioxidant genes Nqo1, Nrf2, and HO-1 and the DNA damage response gene p21 in 14-week old Xpg−/− and wt cerebellum tissue. All values are corrected for TubG2 expression and normalized to wt expression levels; n = 4 animals/group. (F) Quantification of TUNEL-positive cells per mm2 in retinal sections of 4- and 14-week old Xpg−/− and wt mice; n = 6 animals/group. Arrows indicate positive cells. Scale bars: 250 µm (A), 50 µm (B), 100 µm (C), 25 µm (D, F). Error bars indicate standard error of the mean. *p<0.05, **p<0.01.
Figure 5.
Aging features observed in the liver of liver-specific Xpg knockout mice.
(A) Average body weight of C57Bl6/FVB F1 hybrid wild type (wt) males (black triangles), wt females (black circles), liver specific XPG-deficient (Alb-Xpg) males (gray triangles) and Alb-Xpg females (grey circles); n = 4 males/group, n = 2 females/group. (B) Average nucleus size of hepatocytes in the liver of 26- and 52-week old Alb-Xpg and wt mice; n = 4 animals/group. Bottom right: magnification of a nuclear inclusion found regularly in liver sections of 26- and 52-week old Alb-Xpg mice. (C) Quantification of p53-positive cells per cm2 in the liver of 26- and 52-week old Alb-Xpg and wt mice; n = 3 animals/group. (D) Quantification of TUNEL-positive cells per cm2 in the liver of 26- and 52-week old Alb-Xpg and wt mice; n = 3 animals/group. (E) Quantification of Ki67-positive cells per mm2 in the liver of 26- and 52-week old Alb-Xpg and wt mice; n = 3 animals/group. (F) Relative mRNA expression levels of several antioxidant genes and the DNA damage response gene p21 in liver tissue of 26-week old Alb-Xpg and wt mice. All values are corrected for TubG2, Hprt, and Rps9 expression and normalized to wt expression levels; n = 3 animals/group. (G) Relative expression levels of the somatotrophic genes Ghr, Igf1r and Igf1 in liver tissue of 26-week old Alb-Xpg and wt mice. All values are corrected for TubG2, Hprt, and Rps9 expression as internal standard and normalized to wt expression levels; n = 3 animals/group. Scale bars: 25 µm (B, C, D). Error bars indicate standard error of the mean. *p<0.05, **p<0.01.
Figure 6.
Age-related increase of neuronal stress in forebrain-specific Xpg knockout mice.
(A) Average body weight of C57Bl6/FVB F1 hybrid wild type (wt) females (black circles) and forebrain-specific XPG-deficient (Emx1-Xpg) females (gray circles); n≥4 animals/group. (B) Onset of clasping of the hind limbs in Emx1-Xpg mice; n = 7 animals/group. (C) Representative images of GFAP immunostained sagittal neocortex sections of 26- and 52-week old Emx1-Xpg and wt mice showing progressive astrocytosis in Emx1-Xpg mice. (D) Representative images of Mac2 immunostained sagittal brain sections of 26- and 52-week old Emx1-Xpg and wt mice showing Mac2-positive microgliosis and a progressive decrease in size of the cerebral cortex and hippocampus of Emx1-Xpg mice. Arrows indicate microgliosis in corpus callosum and fimbria fornix. A thionin counterstaining was used. (E) Quantification of p53-positive cells in neocortex and cerebellum of 26- and 52-week old Emx1-Xpg and wt mice. Values are the average of four sections per genotype. Arrows indicate p53 positive cells. (F) Representative confocal images showing double labeled p53-NeuN cells in the neocortex (left) and p53-S100ß in the fimbria fornix (right) of 26-week old Emx1-Xpg mice. Arrows indicate p53 positive cells. NCx: neocortex, cc: corpus callosum, Str: striatum, ff: fimbria fornix, Hip: hippocampus. Scale bars: 50 µm (C), 500 µm (D), 200 µm (E) and 20 µm (F). Error bars indicate standard error of the mean. **p<0.01.
Table 2.
Comparison of the Xpg−/− phenotype to that of XP/CS patients.