Fig. 1. Expression of Pds5B and generation of Pds5B-deficient mice. (A) Quantitative RT-PCR analysis of Pds5B mRNA levels in adult mouse tissues normalized to 18S RNA. (Pr, prostate; Te, testis, St, stomach; Lu, lung; Sp, spleen; He, heart; Br, brain; Li, liver; Ki, kidney; Th, thymus). Error bars represent ± s.e.m. (B,C,D) Radioactive in situ hybridization (ISH) using ³⁵S-CTP-labeled probes was performed using WT adult testis (B), adult brain (C), and E13.5 embryo (D). Silver grains indicate Pds5B mRNA. The green background is the Eosin counterstain visualized by darkfield microscopy. Note the high signal at the periphery of the seminiferous tubules (ST) consistent with Pds5B expression in immature germ cells (arrows in B), in hippocampus (arrow in C) and dentate gyrus (arrowhead in C), and wide spread expression in the E13.5 mouse embryo (D). In D, the sagittal sections are probed with antisense (AS, left) and sense control (S, right) probes. (E) Schematic representation of homologous recombination between Pds5B gene (top) and the targeting construct (middle) to give the mutant Pds5B allele (bottom). The coding region of the second exon of Pds5B is replaced by a ß-gal/PGK-Neo (ßGal Neo) cassette. The floxed PGK-Neo cassette was later excised by mating to ß-actin Cre mice. K, KpnI; N, NheI; S, SacI. Black bars with numbers indicate exons and P1-P3 indicate positions of primers used for genotyping. (F) Southern blot analysis depicting successful targeting of the Pds5B locus using genomic DNAs from tails of wild-type (+/+), heterozygous (+/-), homozygous (-/-) mice. The DNA samples were digested with NheI and hybridized with the radiolabeled DNA probe, indicated in E. (G) PCR genotyping analysis of the Pds5B mutation was performed with primers P1, P2, and P3 indicated in E. (H) Relative amounts of Pds5B mRNA from wild-type (+/+), heterozygous (+/-) and homozygous (-/-) embryonic E14.5 brains using qRT-PCR normalized to GAPDH mRNA level. Error bars represent the s.e.m. ^*P<0.001, Student's unpaired t-test. (I) Western blot on E14.5 forelimb tissue lysates was probed with antibodies to PDS5B and ß-tubulin. Note undetectable PDS5B protein in Pds5B^-/- lysate.