Fig. 1. Generation of Af17^-/- mice.

A. Diagrams showing the WT and targeted Af17 loci. An Af17-specific gene trap ES clone (GenBank# CW988924) was used for generation of Af17^-/- mice. In these cells, the gene trap vector carrying βgeo reporter was inserted into intron 7 as shown. The exact insertion site was found 257 bp downstream from the start of intron 7 (GenBank # GU271153). The relative location and orientation of each primer used for genotyping are also indicated. B. A representative agarose gel image showing PCR-based genotyping of genomic DNA. PCR was conducted with primers indicated to amplify a 373-bp WT or 542-bp mutant fragment in separate reactions. C. A representative agarose gel image confirming the disruption of Af17 mRNA. Total RNA was isolated from adult WT and mutant brains and analyzed by RT-PCR in the absence or presence of the reverse transcriptase (RT), with the primers indicated, to amplify 498-bp WT or 467-bp mutant cDNA fragments in separate reactions. β-actin was used as a control of the RT-PCR conditions. D. A representative immunoblot confirming the expression of the Af17-βgeo fusion protein. Total kidney, brain, or heart lysates from four WT (n=4 mice) and three (n=3 mice) mutant mice were prepared. Samples from the same tissue of the same genotype were combined and analyzed by immunoblotting with a chicken antibody recognizing β-galactosidase (Abcam). NS: non-specific.