PMC:1584416 / 5826-9843 JSONTXT

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The corresponding hypomorphic, mutant allele was thus designated as homozygous lethal (†XPCS). Homozygous lethality of the XPCS allele is likely due to reduced levels of expression of this essential protein as a result of gene targeting (Figure 1A) rather than to the mutation itself. Xpd ablation (XpdKO /KO ) is similarly incompatible with life beyond the earliest stages of embryogenesis [22]. Consistent with this interpretation, a different targeted Xpd mutation encoding XPDR683W, which is associated with XP in the homozygous state in humans, was similarly underexpressed and lethal in the homozygous state (designated as †XP allele) (Figure 1A–1C; Table 1; unpublished data). Also, a different targeting approach leading to the use of the native 3′UTR and removal of the neo gene resulted in normalisation of XpdXPCS mRNA levels and viable homozygous XpdXPCS/XPCS (XPDG602D/G602D) animals [23].\nFigure 1 Targeting of the Mouse Xpd Gene\n(A) Schematic representation of the genomic structure and partial restriction map of the wt and targeted mouse Xpd loci. For the wt Xpd allele, shaded boxes represent coding regions of exons 12 and 19–23; the 3′UTR is represented by an open box. TGA indicates the translational stop codon; PolyA indicates the polyadenylation signal. For the XpdTTD targeted allele, the 194–base pair (bp) human XPD cDNA fragment fused to exon 22 is indicated as a striped box including the TTD (R722W) mutation indicated by a vertical arrow. Chicken β-globin exons 2 and 3 including the 3′UTR are indicated as black boxes with corresponding Roman numerals followed by the β-globin polyadenylation signal (PolyA*). For the Xpd†XP and Xpd†XPCS targeted alleles, vertical arrows indicate XPCS (G602D-encoding) and XP (R683W-encoding) mutations in exons 19 and 22, respectively. The unique 3′ probe located outside the targeting construct is marked by a thick black line. Restriction sites: B, BamHI; C, ClaI; E, EcoRI; H, HindIII; Hp, HpaI; Sf, SfiI.\n(B) Southern blot analysis of EcoRI-digested genomic DNA from wt, Xpd†XPCS/wt, and Xpd†XP/wt recombinant embryonic stem cell clones hybridised with the 3′ probe depicted in (A). The wt allele yields a 6.5-kilobase (kb) fragment, whereas both targeted Xpd†XP and Xpd†XPCS alleles yield a 5.1-kb fragment.\n(C) Genotyping of wt and targeted alleles by PCR using primers F2, R1, and mR as indicated in (A) yields fragments of 399 bp and 468 bp, respectively.\n(D) RT-PCR detection of mRNA expression originating from the targeted †XP and †XPCS alleles in embryonic stem cell clones using primers F1 (hybridising outside the targeting construct) and mR as indicated in (A) results in a 1,416-bp fragment.\n(E) Northern blot analysis of total RNA isolated from testis of homozygous wt and XpdTTD/TTD, heterozygous Xpd†XPCS/wt and XpdTTD/wt, and compound heterozygous Xpd†XPCS/TTD mice as indicated. Hybridisation with a 1.4-kb mouse Xpd cDNA probe detects mRNAs of 4, 3.3, and 2.7 kb from wt, Xpd†XPCS, and XpdTTD alleles, respectively. An ethidium bromide (EtBr)–stained gel showing the amount of total RNA loaded is shown below.\nTable 1 Frequency of Xpd†XP/†XP, Xpd†XPCS/†XPCS, and Compound Heterozygous Xpd†XP/†XPCS Embryos and 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