Figure 1.

Generation of Irf8 knock-in (KI) mice. (A) Schematic illustration of the G388S mutation in the mouse Irf8 gene. Displayed at the top is the structure of the mouse Irf8 gene, with introns represented by lines and the direction of transcription indicated by arrows. Exons are depicted as solid boxes, with coding regions as wide boxes and untranslated regions (UTRs) as narrow boxes. Directly beneath the gene structure is a diagram illustrating important domains of the IRF8 protein. The wild-type (WT) and mutant DNA sequences are listed at the bottom, with the CRISPR single-guide RNA (sgRNA) binding region underlined and the PAM shown in bold. The altered amino acid and its codon are shown in red, while a silent mutation is shown in blue. The silent mutation does not result in amino acid change, but it can facilitate the use of polymerase chain reaction (PCR) to differentiate the WT and KI alleles. (B) Sanger sequencing validated G388S knock-in allele in F2 mice. Left panel shows chromatograms of WT sequence. Right panel shows mutant reads. Gray shaded region indicates amino acid(s) at position 388 for each mouse. Arrow indicates desired knock-in mutation (G388S). (C) PCR of genomic DNA with primers flanking exons 2 and 3 shows that WT mice have 1 WT band (365-bp PCR product), heterozygous mice carry a WT band (365 bp) and 2 mutant bands (205 and 160 bp), and homozygous-null mice carry 2 mutant bands (205 and 160 bp). (D) Photographic images of representative WT, Irf8 Het, and Homo mice aged 9 wk. (E) Photographic images for spleen from Irf8 KI WT, Het, Homo, and global knockout mice. (F) Quantitative reverse transcription PCR analysis of Irf8 gene expression in bone marrow macrophages. The data are presented as the mean ± SD, and Het and Homo mice were compared against WT mice. One-way analysis of variance and post hoc Tukey’s test was used for comparisons among groups.