Disruption of the cdk5rap2 gene in DT40 cells. (A) Schematic representation of the gene-targeting strategies. The CNN1 domain maps to exons 2–4 of cdk5rap2 (blue bars). Exons 3–5 (black) were replaced by antibiotic-resistance cassettes. Antibiotic-resistance cassettes flanked by lox sites (triangles) were removed by Cre recombinase (cnn1^lox). The CNN2 domain maps to exons 42–44 of cdk5rap2 (green bars). Exons 41–43 (black) were replaced by antibiotic-resistance cassettes flanked by in-frame STOP codons. The same targeting constructs were introduced into cnn1^lox cells to create the cnn1^loxcnn2^−/− cells. (B) Western blots (wb) of wt and mutant cells. α-Tubulin serves as the loading control. Bands marked by asterisks are nonspecific because in situ tagging of CDK5RAP2 does not give rise to bands of these sizes (see D). (C) Subcellular localization of wt CDK5RAP2 and ΔCNN2. Blue, DNA; red, CDK5RAP2; green, γ-tubulin. (D) Western blot of wt, cnn1^lox, tag-wt, and tag-cnn1^lox cell extracts. tag-wt and tag-cnn1^lox cells contain a protein G–encoding tag in one cdk5rap2 allele. α-Tubulin serves as the loading control. (E) Subcellular localization of tag-CDK5RAP2 and tag-ΔCNN1. Blue, DNA; red, protein G; green, γ-tubulin. (F) Summary of the mutant cdk5rap2 alleles generated. Not known means that these truncated protein products cannot be tracked because of disrupted antibody recognition sites. Percentages in brackets refer to the amount of protein at the centrosome relative to wt. Bars, 5 µm.