• C3a is an anaphylatoxin released during activation of the complement system. The protein encoded by this gene is an orphan G protein-coupled receptor for C3a. The binding of C3a by the encoded receptor activates chemotaxis, granule enzyme release, superoxide anion production, and bacterial opsonization.
• Gene targeting strategy for B-hC3AR1 mice. The exons 1-2 of the mouse C3ar1 gene that encode the whole molecule (ATG to STOP codon), including 3’UTR, were replaced by human counterparts in B-hC3AR1 mice. The promoter and 5’UTR of the mouse gene are replaced by human counterparts. The human C3AR1 expression is driven by the human C3AR1 promoter, while the mouse C3ar1 gene transcription and translation will be disrupted.
• Mouse C3ar1 was detectable in wild-type mice but not in homozygous B-hC3AR1 mice. Human C3AR1 mRNA was detectable in homozygous B-hC3AR1 mice but not in wild-type mice.

Gene targeting strategy for B-hC3AR1 mice. The exons 1-2 of the mouse C3ar1 gene that encode the whole molecule (ATG to STOP codon), including 3’UTR, were replaced by human counterparts in B-hC3AR1 mice. The promoter and 5’UTR of the mouse gene are replaced by human counterparts. The human C3AR1 expression is driven by the human C3AR1 promoter, while the mouse C3ar1 gene transcription and translation will be disrupted.

Strain specific analysis of C3AR1 mRNA expression in wild-type C57BL/6JNifdc mice and B-hC3AR1 mice by RT-PCR. Spleen RNA were isolated from wild-type C57BL/6JNifdc mice (+/+, 12-week-old) and homozygous B-hC3AR1 mice (H/H, 12-week-old), then cDNA libraries were synthesized by reverse transcription, followed by PCR with human and mouse C3AR1 primers. Mouse C3ar1 was detectable in wild-type mice but not in homozygous B-hC3AR1 mice. Human C3AR1 mRNA was detectable in homozygous B-hC3AR1 mice but not in wild-type mice, the sequences were confirmed via Sanger Sequencing.

Strain specific C3AR1 expression analysis in homozygous B-hC3AR1 mice by flow cytometry. Bone marrow cells were collected from C57BL/6JNifdc (+/+, 6-week-old) and homozygous B-hC3AR1 mice (H/H, 6-week-old), then analyzed by flow cytometry with human-specific anti-human C3AR antibodies (Biolegend, 345805). Human C3AR1 was exclusively detectable in homozygous B-hC3AR1 mice but not in wild-type mice.

Strain specific C3AR1 expression analysis in homozygous B-hC3AR1 mice by flow cytometry. Bone marrow cells were collected from C57BL/6JNifdc (+/+, 6-week-old) and homozygous B-hC3AR1 mice (H/H, 6-week-old), then analyzed by flow cytometry with human-specific anti-human C3AR antibodies (Biolegend, 345805). Human C3AR1 was exclusively detectable in homozygous B-hC3AR1 mice but not in wild-type mice.

Strain specific C3AR1 expression analysis in homozygous B-hC3AR1 mice by flow cytometry. Bone marrow cells were collected from C57BL/6JNifdc (+/+, 6-week-old) and homozygous B-hC3AR1 mice (H/H, 6-week-old), then analyzed by flow cytometry with human-specific anti-human C3AR antibodies (Biolegend, 345805). Human C3AR1 was exclusively detectable in homozygous B-hC3AR1 mice but not in wild-type mice.

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6JNifdc mice (female, n=3, 8-week-old) and homozygous B-hC3AR1 mice (female, n=3, 8-week-old). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Percentages of T cells, B cells, NK cells, dendritic cells, neutrophils, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Tregs in B-hC3AR1 mice were similar to those in C57BL/6JNifdc mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.001.