Generation of PGRN-deficient mice. (A) Mouse pgrn locus, targeting vector, and predicted floxed and deleted alleles. Gray boxes denote exons. Sequences are indicated for the neomycin/kanamycin resistance gene (neo/kan; light blue), diphtheria toxin/ampicillin gene (DTX/Amp; pink), LoxP sites (blue triangles), and flippase recombination target sites (red triangles). The red bar marks the 3′ probe. (B) Genotyping of PGRN-deficient mice after crossing PGRN floxed mice with CAG-Cre mice. Genomic DNA was restricted with ScaI for Southern blotting with the 3′ probe. Het, heterozygous; KO, homozygous knockout. (C) PGRN expression monitored by RT-PCR compared with GAPDH as a control (left). Quantification of PGRN transcripts in brain by real-time RT-PCR is shown (right). Results are means ± SEM. B, brain; In, intestine; K, kidney; Li, liver; Sk, skin; Sp, spleen. (D) Hippocampal (top) and cortical (bottom) sections from brains of WT and PGRN-deficient (KO) mice were stained with antibody against PGRN. Bar, 200 µm. (E) Mature BMDMs from 2-mo-old WT or PGRN-deficient mice (KO) were cultured in serum-free media for 48 h. Protein contents of conditioned media were blotted with anti-PGRN (right). Ponceau S staining of the same membrane is shown (left).