Gene targeting strategy for B-hCFB mice. 
The exons 1~18 of mouse Cfb gene that encode the full-length protein were replaced by human CFB exons 1~18 in B-hCFB mice.
The promoter, 5’UTR and 3’UTR region of the mouse gene are retained.

Strain specific analysis of CFB gene expression in wild-type mice and B-hCFB mice by RT-PCR. Mouse Cfb mRNA was detectable in liver of wild-type mice. Human CFB mRNA was detectable only in homozygous B-hCFB mice but not in wild-type mice.

Strain specific FBa expression analysis in homozygous B-hCFB mice by ELISA. Serum were isolated from homozygous B-hCFB mice (H/H) and analyzed by ELISA with FBa ELISA kit (Quidel). FBa was detectable in homozygous B-hCFB mice. (Note: Co-validation data from a client).

H&E staining of B-hCFB mice. 
Kidney tissues from male C57BL/6 and B-hCFB mice (n=3, 7-week-old) were collected and analyzed for H&E staining. There were no visible lesions in either measurement between C57BL/6 and B-hCFB mice, indicating that the introduction of hCFB in place of its mouse counterpart does not change the health of the kidney.

Alternative complement pathway activity on rabbit erythrocytes. Serum collected from homozygous B-hCFB mice (male, n=3 serum mix) were measured OD415 on rabbit erythrocytes. The alternative pathway could be rescued by adding human C3b+ C5 and C3+ C5 recombinant protein in vitro. 
(Note: Co-validation data from client).

Alternative complement pathway activity. Serum collected from homozygous B-hCFB mice (male, 5-6w). The alternative pathways assay using ELISA kit (Hycult Biotech, HIT422) with LPS coated plate and detecting C9 in MAC/TCC complex (information from the support of the HIT422). The alternative pathway could be rescued by adding human C3b+C5, and C3+C5 recombinant protein in vitro. Values are expressed as mean ± SEM.

The inhibitory efficiency of the nucleic acid drugs against human CFB in B-hCFB mice. B-hCFB mice were randomly divided into three groups (n=3/group, 8 weeks old, male). The human CFB targeted siRNA-analog synthesized according to patents (G2 1mpk, G3 3mpk) and G1 PBS were administered to the mice individually. The mice were sacrificed on day 7, the liver tissue and plasma were collected to detect the expression level of human CFB mRNA by qPCR and protein by ELISA . (A) The schematic diagram of experimental processing. (B) The % human CFB protein remaining of B-hCFB mice. (C) The expression of human CFB mRNA in liver. The human CFB in the treatment group (G2, G3) was significantly reduced compared to the control group (G1). Values are expressed as mean ± SEM. Significance was determined by a one-way ANOVA test. ****p < 0.0001.

Blood chemistry tests of B-hCFB mice. 
Serum from male C57BL/6 and B-hCFB mice (n=6, 7 week-old) was collected and analyzed for levels of UREA, CREA, and TP. There were no differences in either measurement between C57BL/6 and B-hCFB mice, indicating that the introduction of hCFB in place of its mouse counterpart does not change UREA, CREA, and TP levels or the health of kidney. Values are expressed as mean ± SEM.