• The official symbol of mouse C5 in NCBI is Hc. This C5 gene encodes a component of the complement system. It's part of the innate immune system and important for inflammation, host homeostasis and defense against pathogens. The encoded preproprotein generates multiple products like the C5 alpha chain, beta chain, C5a anaphylatoxin, and C5b. C5 protein consists of alpha and beta chains linked by a disulfide bridge. Cleavage of the alpha chain forms C5a anaphylatoxin with spasmogenic and chemotactic activity and C5b. Mutations cause complement component 5 deficiency with recurrent bacterial infections.
• Gene targeting strategy: The exons 1-39 of the mouse Hc gene were replaced by exons 1-41 of the human C5 gene that encode the whole molecule (ATG to STOP codon), including 5’UTR and 3’UTR in B-hC5 mice plus. The human C5 expression was driven by human C5 promoter, while the mouse Hc gene transcription and translation will be disrupted.
• mRNA expression analysis: Mouse C5 mRNA was detectable only in liver of wild-type C57BL/6JNifdc mice (+/+). Human C5 mRNA was exclusively detectable in homozygous B-hC5 mice plus (H/H) but not in wild-type mice.
• Protein expression analysis: Mouse C5 and C5a was detectable only in liver of wild-type C57BL/6JNifdc mice (+/+). Human C5 and C5a was exclusively detectable in homozygous B-hC5 mice plus (H/H) but not in wild-type mice.
• In vitro functional analysis: The alternative complement pathway was observed, and the LNP023 can inhibit the alternative complement pathway activity both in wild-type mice and B-hC5 mice plus.
• Application: This model is useful for investigating the efficacy of alternative complement pathway-related diseases. And this model can be used for preclinical pharmacodynamics studies of small nucleic acid drugs.

Gene targeting strategy for B-hC5 mice plus. The exons 1-39 of the mouse Hc gene were replaced by exons 1-41 of the human C5 gene that encode the whole molecule (ATG to STOP codon), including 5’UTR and 3’UTR in B-hC5 mice plus. The human C5 expression was driven by human C5 promoter, while the mouse Hc gene transcription and translation will be disrupted.

Strain specific analysis of C5 mRNA expression in wild-type C57BL/6JNifdc mice and homozygous B-hC5 mice plus by RT-PCR. Liver RNA were isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hC5 mice plus (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse and human C5 primers. Mouse C5 mRNA was detectable only in liver of wild-type C57BL/6JNifdc mice (+/+). Human C5 mRNA was exclusively detectable in homozygous B-hC5 mice plus (H/H) but not in wild-type mice.

Strain specific C5 expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hC5 mice plus by ELISA. Serum was collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hC5 mice plus (H/H) (male, 8 weeks old, n=3). Expression levels of mouse and human C5 were analyzed by ELISA (anti-mouse C5 ELISA kit: abcam, ab264609; anti-human C5 ELISA kit: abcam, ab125963). Mouse C5 was only detectable in wild-type C57BL/6JNifdc mice. Human C5 was exclusively detectable in homozygous B-hC5 mice plus. Values are expressed as mean ± SEM.

Strain specific C5a expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hC5 mice plus by ELISA. Serum was collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hC5 mice plus (H/H) (male, 8 weeks old, n=3). Expression levels of mouse and human C5a were analyzed by ELISA (anti-mouse C5a ELISA kit: Abcam, ab193718; anti-human C5a ELISA kit: Abcam, ab193695). Mouse C5a was only detectable in wild-type C57BL/6JNifdc mice. Human C5a was exclusively detectable in homozygous B-hC5 mice plus. Values are expressed as mean ± SEM.

In vitro functional analysis of the alternative pathway. Serum was collected from wild-type C57BL/6JNifdc mice (+/+), homozygous B-hC5 mice plus (H/H), and B-Cfh KO mice (-/-) (male, 8 weeks old, n=1-2) and evaluated the functional activity with the complement kit (Alternative Complement Pathway, Mouse Assay, Hycult Biotech, HIT422). LNP023 is an inhibitor of the alternative complement pathway by targeting factor B (1μM, 15 min, RT). Serum was pre-treated with LNP023 or not. (A) The OD value in 20% serum dilutions. (B) Alternative pathway activity compared with wild-type mice. B-Cfh KO mice were the negative control, since the deficiency of CFH aggravates the cleavage of C3 in serum, leading to a decrease in the alternative pathway activity.