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. 2021 Jun 21;10:e67290. doi: 10.7554/eLife.67290

Figure 2. Sp140–/– mice are susceptible to bacterial pathogens.

(A) RT-PCR of cDNA from BMMs of the indicated genotypes. Red arrow indicates band corresponding to a portion of Sp140, verified by sequencing. (B) Immunoblot of lysates from Sp140–/– and B6 BMMs treated with 10 U/ml of recombinant mouse IFNγ for 24 hr. Equal amounts of protein were loaded for immunoblot with anti-SP140 antibody. (C–F) Mice were infected with Mycobacterium tuberculosis and measured for (C) lung CFU at 28 days post-infection, (E) body weight over time, and (F) survival. Statistics in (E) shows the comparison to B6 at day 28, and data are from 10 B6, 11 B6.Sst1S, 11 Sp110–/–, 14 Sp140–/–, and 6 Sp140+/–mice. (D) H&E staining of lungs at 25 days post-infection with M. tuberculosis. Full histology images are provided in Figure 2—figure supplement 2. (G) Mice were infected with Legionella pneumophila and lung CFUs were determined at 96 hr post-infection. All mice were bred in-house, Sp140–/– and Sp140+/– were littermates (C–F). (C), (E), and (G) are combined results of two independent infections. (A–D) show representative analysis of one Sp140–/– line (line 1), whereas (F, G) include a mixture of both lines 1 and 2. Results of infection of both lines with M. tuberculosis are shown in Figure 2—figure supplement 1E. (C, E, F, G) Mann-Whitney test. *p≤0.05; **p≤0.01; ***p≤0.005. BMM, bone marrow-derived macrophage; CFU, colony-forming unit; H&E, hematoxylin and eosin; RT-qPCR, real-time quantitative-polymerase chain reaction; WT, wild-type.

Figure 2.

Figure 2—figure supplement 1. CRISPR/Cas9 targeting strategy for Sp140–/– and validation of founders.

Figure 2—figure supplement 1.

(A) Mouse Sp140 gene. Guide RNA sequence for CRISPR/Cas9 targeting and protospacer-adjacent motif (PAM) are indicated. (B, C) Sp140 locus in wild-type (WT) and two independent founders of Sp140–/– validated by sequencing. (D) Immunoblot for SP110 using BMMs from mice of the indicated genotypes. Intervening lanes have been removed for clarity (indicated by line in the image). (E) Mycobacterium tuberculosis-infected mice were harvested for CFU at 25 days post-infection. Empty and filled triangles indicate the two independent lines of Sp140–/– used in this infection. All mice were bred in-house and Sp140+/– ± littermates with Sp140–/– line 2. Mann-Whitney test. *p≤0.05; **p≤0.01; ***p≤0.005. BMM, bone marrow-derived macrophage; CFU, colony-forming unit.
Figure 2—figure supplement 2. Histology of lungs from B6, B6.Sst1S, Sp110–/–, and Sp140–/– mice after infection with Mycobacterium tuberculosis.

Figure 2—figure supplement 2.

H&E staining of entire lung sections from mice of indicated genotypes at 25 days post-infection with M. tuberculosis. Black squares denote sections shown in Figure 2C. Each image represents a lung section from a different mouse. Borders in background color have been added around each image. Scale bar applies to all images. Samples were evaluated and scored (0–4, least to most) for macrophage (histocyte), lymphoid, granulocyte infiltration, and extent of necrosis. H&E, hematoxylin and eosin.
Figure 2—figure supplement 3. Characterization of off-target genes mutated in Sp140–/– mice.

Figure 2—figure supplement 3.

(A) Schematic of amplicon sequencing strategy for Sp140 and Sp140 homologs. (B) Summary of edited Sp140 homologs from amplicon sequencing and RNA-seq analysis. SNPs are denoted based on the Sp140 X1 transcript. Expression level was roughly estimated from read counts. Three B6 and two Sp140–/– mice from each founder line were used as biological replicates for Sp140 exon 2/3 amplicon sequencing from cDNA, two mice per genotype were used for Sp140 exon 3 amplicon sequencing from cDNA, and one mouse per genotype was used for Sp140 exon 3 amplicon sequencing from DNA. SNP, single-nucleotide polymorphism.
Figure 2—figure supplement 4. Complementation of hyper type I IFN responses in Sp140–/– BMMs.

Figure 2—figure supplement 4.

(A) BMMs were left untreated or treated with TNF-α for 24 hr. Total RNA was used for RT-qPCR. Averages of technical duplicates for one biological replicate are shown. Data is representative of two independent experiments. (B) RT-qPCR of Sp140–/– BMMs transduced with either control SINV-minCMV-GAL4-mNeonGreen (SINV-mNeonGreen) or SINV-minCMV-Sp140 (SINV-Sp140), primed with 5 ng/mL IFN-𝛾 for 14 hr and treated with 10 ng/mL TNF-⍺ for 4 hr. *p≤0.05 calculated with an unpaired t-test with Welch’s correction. Data are representative of two independent experiments. BMM, bone marrow-derived macrophage; RT-qPCR, real-time quantitative-polymerase chain reaction.