Generation and confirmation of a <i>creERT2</i> knock-in allele of <i>Upk3b</i>.

<p>(A) Scheme of the targeted insertion of a <i>creERT2</i> recombinase gene/<i>loxP</i>-flanked <i>neomycin</i> selection cassette in the <i>Upk3b</i> locus. Exons are shown in black, regions for homologous recombination in red. Screening for clones with correct integration of the <i>creERT2/neo</i> cassette was performed using a PCR for the 3′-region, primers are indicated in pink. (B) A <i>Bam</i>HI restriction fragment length polymorphism (RFLP) with the indicated 5′-probe was used to check for correct 5′-integration, wildtype (wt) and mutant (mut) bands are shown on the Southern blot. (C) A long-range PCR was used to verify the correct 3′-integration of the targeting vector by detection of a 3.5 kbp fragment in the mutant allele. ATG, transcriptional start codon; B, <i>Bam</i>HI; <i>creERT2</i>, <i>cre</i> recombinase fused to a triple mutant form of the human estrogen receptor expression cassette; kbp, kilo base pairs; <i>loxP</i>, locus of X-over P1; Neo, <i>neomycin</i> resistance gene; pA, polyadenylation signal; pgk, phosphoglycerate kinase I promoter.