Description

• VSIG4 is expressed in macrophages but not in monocytes. It binds to the β-chain of C3b and C3c and inhibits the AP C3 and C5 convertases.
• LAIR1 is expressed on T cells, B cells, NK cells, monocytes, macrophages and neutrophils. LAIR1 is also expressed on tumor cells. It binds to collagen and C1q and inhibits the activation or differentiation of these cells.
• The coding sequences of human VSIG4 and mouse 3’UTR were inserted into exon 2 of mouse Vsig4 gene in B-hVSIG4 mice. The exons 3-4 of mouse Lair1 gene that encode the extracellular domain were replaced by human LAIR1 exons 3-6 in B-hLAIR1 mice. B-hVSIG4/hLAIR1 mice was obtained by crossing B-hVSIG4 mice and B-hLAIR1 mice together.
• Human VSIG4 was exclusively detectable in PEMs of homozygous B-hVSIG4/hLAIR1 mice by FACS. Human LAIR1 was detectable in T cells, B cells, NK cells, monocytes, macrophages and neutrophils of homozygous B-hVSIG4/hLAIR1 mice by FACS.
• Application: B-hVSIG4/hLAIR1 mice is used for pharmacodynamics and safety evaluation of drugs targeting to human VSIG4 and/or LAIR1 in mouse tumor or autoimmune disease models.

Targeting Strategy

Gene targeting strategy for B-hVSIG4/hLAIR1 mice.

The coding sequences of human VSIG4 and mouse 3’UTR were inserted into exon 2 of mouse Vsig4 gene in B-hVSIG4 mice. The promoter and 5’UTR region of the mouse gene are retained. The humanized LAIR1 expression is driven by endogenous mouse LAIR1 promoter, while mouse LAIR1 gene transcription and translation will be disrupted.

The exons 3-4 of mouse Lair1 gene that encode the extracellular domain were replaced by human LAIR1 exons 3-6 in B-hLAIR1 mice. The genomic region of mouse LAIR1 gene that encodes the signal peptide, transmembrane and cytoplasmic domain are retained. The promoter, 5’UTR and 3’UTR region of the mouse gene are retained. The chimeric LAIR1 expression is driven by endogenous mouse LAIR1 promoter, while mouse LAIR1 gene transcription and translation will be disrupted.

B-hVSIG4/hLAIR1 mice was obtained by crossing B-hVSIG4 mice and B-hLAIR1 mice together.

Protein Expression Analysis in PEMs-VSIG4

Strain specific VSIG4 expression analysis with peritoneal exudate macrophages (PEMs) from homozygous B-hVSIG4/hLAIR1 mice by flow cytometry. PEMs were collected from wild-type C57BL/6N (+/+) and homozygous B-hVSIG4/hLAIR1 mice (H/H), and analyzed by flow cytometry with species-specific anti-mouse VSIG4 antibody (eBioscience, 12-5752-82), and anti-human VSIG4 antibody (eBioscience, 17-5757-41). Mouse VSIG4 was only detectable in wild-type mice. Human VSIG4 was exclusively detectable in homozygous B-hVSIG4/hLAIR1 mice.

Protein Expression Analysis in Spleen-LAIR1

Strain specific LAIR1 expression analysis in homozygous B-hVSIG4/hLAIR1 mice by flow cytometry. Splenocytes cells were collected from wild-type C57BL/6N (+/+) and homozygous B-hVSIG4/hLAIR1 mice (H/H), and analyzed by flow cytometry with species-specific anti-mouse LAIR1 antibody (eBioscience, 12-3051-81), and anti-human LAIR1 antibody (Biolegend, 342802). Mouse LAIR1 was only detectable in wild-type mice. Human LAIR1 was exclusively detectable in homozygous B-hVSIG4/hLAIR1 mice.

Strain specific LAIR1 expression analysis in homozygous B-hVSIG4/hLAIR1 mice by flow cytometry. Splenocytes cells were collected from wild-type C57BL/6N (+/+) and homozygous B-hVSIG4/hLAIR1 mice (H/H), and analyzed by flow cytometry with species-specific anti-mouse LAIR1 antibody (eBioscience, 12-3051-81), and anti-human LAIR1 antibody (Biolegend, 342802). Mouse LAIR1 was only detectable in wild-type mice. Human LAIR1 was exclusively detectable in homozygous B-hVSIG4/hLAIR1 mice.

Protein Expression Analysis in Blood-LAIR1

Strain specific LAIR1 expression analysis in homozygous B-hVSIG4/hLAIR1 mice by flow cytometry. Blood cells were collected from wild-type C57BL/6N (+/+) and homozygous B-hVSIG4/hLAIR1 mice (H/H), and analyzed by flow cytometry with species-specific anti-mouse LAIR1 antibody (eBioscience, 12-3051-81), and anti-human LAIR1 antibody (Biolegend, 342802). Mouse LAIR1 was only detectable in wild-type mice. Human LAIR1 was exclusively detectable in homozygous B-hVSIG4/hLAIR1 mice.

Strain specific LAIR1 expression analysis in homozygous B-hVSIG4/hLAIR1 mice by flow cytometry. Blood cells were collected from wild-type C57BL/6N (+/+) and homozygous B-hVSIG4/hLAIR1 mice (H/H), and analyzed by flow cytometry with species-specific anti-mouse LAIR1 antibody (eBioscience, 12-3051-81), and anti-human LAIR1 antibody (Biolegend, 342802). Mouse LAIR1 was only detectable in wild-type mice. Human LAIR1 was exclusively detectable in homozygous B-hVSIG4/hLAIR1 mice.

Frequency of Leukocyte Subpopulations in Spleen

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6N mice (male, n=3, 12-week-old) and homozygous B-hVSIG4/hLAIR1 mice (male, n=3, 12-week-old). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Frequencies of T cells, B cells, NK cells, dendritic cells, neutrophils, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Tregs in B-hVSIG4/hLAIR1 mice were similar to those in C57BL/6N mice. The frequency of leukocyte subpopulations in blood and lymph nodes of B-hVSIG4/hLAIR1 mice were also comparable to wild-type C57BL/6N mice (Data not shown). Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***P < 0.001.