Fig. 3
Generation of the Iba1-iCre mouse line. a Schema of Iba1 genomic DNA and the targeted genome. Open and filled boxes indicate noncoding and coding exons, respectively. Filled circles in the Iba1iCre allele delineate the 5′ and 3′ termini of the targeting vector. The vector was constructed to insert an improved Cre recombinase gene (iCre), followed by an internal ribosome entry site (IRES) and a FLAG-tagged EGFP, in frame into the translational initiation site of the Iba1 gene. Blue arrows show primer positions for PCR genotyping. Blue bars indicate probes for Southern blot analysis. Two frt sequences (semicircles) are attached to remove the neomycin resistance gene (Neo). E, EcoRV; N, NdeI. b Southern blot analysis for genomic DNAs from wild-type (+/+) and targeted (+/iCre) ES cells. Left, EcoRV-digested genomic DNA hybridized with 5′ probe; middle and right, NdeI-digested DNA hybridized with Neo or 3′ probe, respectively. c Genomic PCR genotyping of Iba1+/iCre intercrosses using primers F, R1 and R2. d Immunoblots of crude fractions from whole mouse brains with anti-Iba1 and actin antibodies. e–i Cre recombinase activity induced by Iba1-iCre in CAG-floxed STOP tdTomato reporter mice. No EGFP fluorescence was detected in the Iba1-iCre cerebellum. Immunostaining for Iba1 (green) and tdTomato fluorescence (red) in lobules IV–V in the vermis of Iba1iCre/+; tdTomato (e–g) and control (Iba1+/+; tdTomato) (h, i) cerebella at P30. All Iba1-positive cells expressed tdTomato, but there were several tdTomato-labeled processes with very weak or no Iba1 immunoreactivity (arrowheads in e–p). j–n Immunostaining for cell markers (green) in Iba1iCre/+; tdTomato mice at P30; tdTomato did not overlap with markers for astrocyte (GFAP, j), oligodendrocyte (APC, k), NG2 chondroitin sulfate proteoglycan (l), neurons (NeuN, m) or inhibitory neurons (parvalbumin, n). o–r Localization of Iba1 and tdTomato signals at P7 (o) or P14 (p). q, r Fractions of tdTomato-labeled cells relative to Iba1-positive cells (q) or Iba1-negative cells relative to tdTomato-labeled cells (r) (n = 310 cells from two mice at P7, n = 191 cells from two mice at P14). A scale bar, 50 μm (e–p). Error bars, SEM
