Targeting of the Mouse <i>Xpd</i> Gene

<div><p>(A) Schematic representation of the genomic structure and partial restriction map of the wt and targeted mouse <i>Xpd</i> loci. For the wt <i>Xpd</i> 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 <i>Xpd^TTD</i> targeted allele, the 194–base pair (bp) human <i>XPD</i> 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 <i>Xpd^†XP</i> and <i>Xpd^†XPCS</i> 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.</p> <p>(B) Southern blot analysis of EcoRI-digested genomic DNA from wt, <i>Xpd^†XPCS/wt,</i> and <i>Xpd^†XP/wt</i> 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 <i>Xpd^†XP</i> and <i>Xpd^†XPCS</i> alleles yield a 5.1-kb fragment.</p> <p>(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.</p> <p>(D) RT-PCR detection of mRNA expression originating from the targeted <i>†XP</i> and <i>†XPCS</i> 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.</p> <p>(E) Northern blot analysis of total RNA isolated from testis of homozygous wt and <i>Xpd^TTD/TTD,</i> heterozygous <i>Xpd^†XPCS/wt</i> and <i>Xpd^TTD/wt,</i> and compound heterozygous <i>Xpd^†XPCS/TTD</i> mice as indicated. Hybridisation with a 1.4-kb mouse <i>Xpd</i> cDNA probe detects mRNAs of 4, 3.3, and 2.7 kb from wt, <i>Xpd^†XPCS,</i> and <i>Xpd^TTD</i> alleles, respectively. An ethidium bromide (EtBr)–stained gel showing the amount of total RNA loaded is shown below.</p>