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B-hC5/hC5AR1 mice
Strain Name C57BL/6-Hctm1(C5)C5ar1tm1(C5AR1)/Bcgen Common Name  B-hC5/hC5AR1 mice
Background C57BL/6 Catalog number  121215
Aliases 
C5: C5Da, C5b, CPAMD4, ECLZB; C5AR1: C5A; C5AR; C5R1; CD88

Gene description


C5 encodes a component of the complement system, a part of the innate immune system that plays an important role in inflammation, host homeostasis, and host defense against pathogens. The encoded preproprotein is proteolytically processed to generate multiple protein products, including the C5 alpha chain, C5 beta chain, C5a anaphylatoxin and C5b. The C5 protein is comprised of the C5 alpha and beta chains, which are linked by a disulfide bridge. Cleavage of the alpha chain by a convertase enzyme results in the formation of the C5a anaphylatoxin, which possesses potent spasmogenic and chemotactic activity, and the C5b macromolecular cleavage product, a subunit of the membrane attack complex (MAC). Mutations in this gene cause complement component 5 deficiency, a disease characterized by recurrent bacterial infections. 
C5AR1 is the receptor for C5a, plays a critical role in numerous inflammatory conditions. C5a is a member of the complement cascade, an important mechanism for host defense against bacteria. Increased complement activation and excessive production of C5a have been implicated in the pathogenesis of several inflammatory diseases, and considerable effort has gone into developing C5aR antagonists, including organic small molecules and peptide antagonists. As one of the most potent mediators of inflammation, the complement component C5a is a promising target for anti-inflammatory drugs. However, a drawback of using animal models to develop antagonists of complement activation is that interspecies differences in receptors and ligands may complicate drug development and delay the evaluation of their efficacy in humans.  We reasoned that the availability of human C5aR knock-in mice might provide an effective means to validate this receptor in mouse models of disease, using reagents that could be developed for use in clinical trials.


mRNA expression analysis

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Strain specific analysis of C5 and C5AR1 gene expression in wild-type C57BL/6 mice and B-hC5/hC5AR1 mice by RT-PCR. Mouse Hc and C5ar1 mRNA were detectable only in lung cells of wild-type C57BL/6 mice (+/+). Human C5 and C5AR1 mRNA were detectable only in homozygous B-hC5/hC5AR1 mice (H/H), but not in wild-type mice. 


Protein expression analysis 

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Strain specific C5 and C5a expression analysis in homozygous B-hC5/hC5AR1 mice by ELISA. Serum was collected from wild-type C57BL/6 mice (+/+; +/+) or homozygous B-hC5/hC5AR1 mice (H/H; H/H) and analyzed by ELISA with anti-C5 and anti-C5a antibodies. A. Mouse C5 and C5a were detectable in wild-type mice. Human C5 and C5a were exclusively detectable in homozygous B-hC5/hC5AR1 mice but not in wild-type mice. B. The concentration of C5 in male mice is higher than that in female mice.

Protein expression analysis

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Strain specific C5AR1 expression analysis in homozygous B-hC5/hC5AR1 mice by FACS. Bone marrow was collected from wild-type C57BL/6 mice (+/+; +/+) and homozygous B-hC5/hC5AR1 mice (H/H; H/H), and analyzed by FACS with anti-C5AR1 antibody. Mouse C5AR1 was only detectable in wild-type mice. Human C5AR1 was exclusively detectable in homozygous B-hC5/hC5AR1 mice but not in wild-type mice.


Analysis of leukocytes cell subpopulation in spleen

from clipboard    Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hC5/hC5AR1 mice (n=3, 6-week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for the CD45+ population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hC5/hC5AR1 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hC5/hC5AR1 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in spleen. Values are expressed as mean ± SEM.


Analysis of T cell subpopulation in spleen

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    Analysis of spleen T cell subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hC5/hC5AR1 mice (n=3, 6-week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ cells were gated for TCRβ+ T cell population and used for further analysis as indicated here. B. Results of FACS analysis. The percent of CD4+ T cells, CD8+ T cells and Tregs in homozygous B-hC5/hC5AR1 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hC5/hC5AR1 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in spleen. Values are expressed as mean ± SEM.


Analysis of leukocytes cell subpopulation in lymph node

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Analysis of lymph node leukocyte subpopulations by FACS. Lymph nodes were isolated from female C57BL/6 and B-hC5/hC5AR1 mice (n=3, 6-week-old). Flow cytometry analysis of the leukocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for the CD45+ population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hC5/hC5AR1 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hC5/hC5AR1 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in lymph node. Values are expressed as mean ± SEM.


    Analysis of T cell subpopulation in lymph node
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Analysis of lymph node T cell subpopulations by FACS. Leukocytes were isolated from female C57BL/6 and B-hC5/hC5AR1 mice (n=3, 6-week-old). Flow cytometry analysis of the leukocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ cells were gated for TCRβ+ T cell population and used for further analysis as indicated here. B. Results of FACS analysis. The percent of CD4+ T cells, CD8+ T cells, and Tregs in homozygous B-hC5/hC5AR1 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hC5/hC5AR1 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in lymph node. Values are expressed as mean ± SEM.


Analysis of leukocytes cell subpopulation in blood

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Analysis of blood leukocyte subpopulations by FACS. Blood cells were isolated from female C57BL/6 and B-hC5/hC5AR1 mice (n=3, 6-week-old). Flow cytometry analysis of the blood cells was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for the CD45+ population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hC5/hC5AR1 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hC5/hC5AR1 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in blood. Values are expressed as mean ± SEM.


Analysis of T cell subpopulation in blood

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Analysis of blood T cell subpopulations by FACS. Blood cells were isolated from female C57BL/6 and B-hC5/hC5AR1 mice (n=3, 6-week-old). Flow cytometry analysis of the blood cells was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ cells were gated for TCRβ+ T cell population and used for further analysis as indicated here. B. Results of FACS analysis. The percent of CD4+ T cells, CD8+ T cells, and Tregs in homozygous B-hC5/hC5AR1 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hC5/hC5AR1 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in blood. Values are expressed as mean ± SEM.