Generation of <i>Xpg^−/−</i> mice.

<p>(A) Genomic organization and disruption strategy for <i>Xpg</i> depicting the wild type allele (<i>+</i>), the targeting construct, the targeted allele (<i>fn</i>), the conditional allele after Flp-mediated recombination of <i>Frt</i> sites (<i>f</i>) and the targeted <i>Xpg</i> allele following subsequent Cre-mediated recombination of <i>LoxP</i> sites (<i>−</i>). 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 (<i>wt</i>) allele yields a 7.4-kb fragment whereas the targeted (<i>tg</i>) 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 <i>wt</i> and <i>tg</i> 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 <i>Xpg^−/−</i> and <i>wt</i> MDFs using a rabbit polyclonal antibody raised against a peptide conserved between human and mouse XPG. Tubulin is used as loading control. (E) Primary <i>Xpg^−/−</i> and <i>wt</i> 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 <i>Xpg^−/−</i> and <i>wt</i> MDFs reveals a severe GG-NER defect in <i>Xpg^−/−</i> cells. MDFs were irradiated with 16 J/m² of UV-C. UDS levels are expressed relative to the non-irradiated <i>wt</i> cells. (G) UV-induced RRS in primary <i>Xpg^−/−</i> and <i>wt</i> MDFs reveals a severe TC-NER defect in <i>Xpg^−/−</i> cells. MDFs were irradiated with 16 J/m² of UV-C. 16 h after UV irradiation the <i>wt</i> cells show recovery of RNA synthesis, while <i>Xpg^−/−</i> MDFs only show residual activity in nucleoli (rRNA transcription). Arrowheads indicate nuclei. Error bars indicate standard error of the mean. **p<0.01.