<i>Azi1^Gt</i> is a null allele of <i>Azi1</i>.

<p>(A) Schematic of the <i>Azi1</i> gene trap inserted in intron 2 of <i>Azi1</i>. Exons are shown as boxes with translated transcript shaded. Arrows indicate primers used to characterise mRNA expression in <i>Azi1^Gt/Gt</i> mice. SA: Splice acceptor, T2A: self-cleaving peptide, pA: polyA. (B) <i>Azi1</i> mRNA expression in kidneys (K) and ovaries (O) from <i>Azi1^+/+</i> and <i>Azi1^Gt/Gt</i> mice was examined. No expression was detected across the gene trap insertion site in <i>Azi1^Gt/Gt</i> mice. (C) The lack of wild type transcription across the gene trap insertion site was confirmed by qPCR on testes cDNA. (D) LacZ staining of E11.5 <i>Azi1^Gt/+</i> embryos demonstrates the gene trap-βGeo cassette is expressed. <i>Azi1</i> expression is ubiquitous during development with higher expression in the limbs, somite derivatives, eyes and brain. (E) No full length or truncated Azi1 protein expression was detected in <i>Azi1^Gt/Gt</i> mutant testes. A strong band was detected in wild type and at lower levels in <i>Azi1^+/Gt</i> testes extract using an antibody raised to the C-terminal of Azi1 (SF91) (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003928#pgen.1003928.s003" target="_blank">Figure S3A</a>). Densitometry was used to give Azi1 levels relative to α-Tubulin, normalised to 1 in wild type (lower of panel D). <i>Azi1^Gt/Gt</i> mice are null for <i>Azi1</i>. (F–H) Endogenous Azi1 is detected around centrosomes of wild type MEFs (F) and multiciliated airway epithelial cells (G and H). No Azi1 expression was detected by immunofluorescent staining of MEFs or nasal brush biopsies from <i>Azi1^Gt/Gt</i> mice, further confirming these mice are null for Azi1. Bottom panel shows the secondary only control. Inset in F highlights the centrosome. Arrowheads in G and H highlight the apical surface where γ-Tubulin positive centrosomes dock, and where Azi1 is localised in wild type cells. Scale bars represent 10 µm (F–H).