Fig. 1

Verification of global deletion of Stap1 gene expression. Panel a shows the gene targeting details for the generation of Stap1^−/− mice. This construct is a ‘knockout-first’, which can be converted to a conditional, upon deletion of the LacZ-Neo cassette. The mice used in this study remain as knockout and retain this cassette. A tissue survey of Stap1 mRNA was performed using qPCR in tissues collected from Stap1^+/+ mice (panel b, n = 2). Note that compared to a house-keeping gene expression (see Methods), only spleen and lung showed some expression with very low levels detected in other tissues. Stap1^−/− mice (KO) showed no detectable qRT-PCR products (panel c, n = 2–3) compared to wild-type mice (WT) in spleen, liver or kidney. To further confirm a lack of legitimate transcript expression, RT-PCR analysis of exon splicing for exons 2–5, 2–6, and 2–7 in liver and spleen was performed (panel d). The labels ‘5, 6, and 7’ for each tissue indicates the location of the reverse primers targeting exons 5, 6, or 7 respectively, used with a forward primer located in exon 2. Spleen mRNA from WT tissues amplified the correct expected size of product and size increments (exon 2–5; 251 bp, exon 2–6; 482 bp, and exon 2–7; 536 bp indicated by filled arrowheads). No products were noted when exons 2–5, or 2–6 were used for KO spleen mRNA. A slightly larger product was noted for exons 2–7 (indicated by open arrowhead), though on sequencing this was found to be a non-specific product from mis-priming (see Text). Error bars denote ±1 SD