Abstract
Factor B is a serine protease, essential for the function of the alternative pathway of complement activation. To study further the importance of the alternative pathway of complement activation in vivo and to help elucidate any additional functions of factor B or its activation fragments we developed, by homologous recombination in embryonic stem cells, mice with a disrupted factor B gene. Factor B-deficient mice produced no detectable factor B mRNA or protein and had no detectable factor B enzymatic activity or alternative pathway function in their serum. Further studies revealed that the two adjacent genes, complement component C2 and D17H6S45, had been down regulated as a result of the disruption. The down-regulation of C2 gene expression was sufficient to cause a complete loss of classical pathway function as determined by the failure of sera from the deficient mice to opsonize antibody-sensitized sheep erythrocytes and by impairment of immune complex processing in vivo. The resulting mouse is deficient in both factor B and C2, and hence the alternative and classical pathways of complement activation, and adds to the repertoire of models for studying the in vivo role of complement in the immune system.
Highlights
The alternative pathway (AP)1 of complement activation forms an ancient part of the innate immune system [1]
The gene lies directly adjacent to the gene for complement component C2, the polyadenylation signal of C2 overlapping with the 5Ј-regulatory region of H2-Bf [3, 4]
The murine H2-Bf gene was disrupted in embryonic stem (ES) cells by homologous recombination with the targeting vector pH2-Bf.lacZ/ neo-herpes simplex virus thymidine kinase gene (HSVtk)
Summary
Mice with a disrupted H2-Bf locus were generated by homologous recombination in ES cells [23] (Fig. 1A). ES cells were transfected by electroporation with the linearized targeting vector pH2-Bf.lacZ/neo-HSVtk. Colonies surviving positive/negative selection [24] were isolated and screened by Southern blot [25] of EcoRI-digested genomic DNA using a 3Ј-external probe (P1) (Fig. 1B). After identification of clones with a disrupted H2-Bf gene a 5Ј-external probe (P2) was used to confirm that genuine homologous recombination had occurred on both sides of the gene. The cDNA from the gene for complement component C4 (C4) was amplified with the primers C4/2ϩ (5Ј-GAAGGACTTTAAGCTGAGCTC-3Ј) and C4/3Ϫ (5Ј-CTGAGCTACCAGCTGGATGTG-3Ј), resulting in a 129-bp product. Total Hemolytic Activity—The classical pathway mediated total hemolytic complement activity in mouse serum was determined by release of 51Cr-labeled hemoglobin from antibody-sensitized sheep erythrocytes as described previously [27]. Alternative Pathway Hemolytic Activity—The protocol for the alternative pathway hemolytic assay was the same as the total hemolytic activity assay except that a 5% v/v suspension of guinea pig erythrocytes in PBS, 0.007 M Mg2ϩ, 0.01 M EGTA (pH 7.2) was used instead of sensitized sheep erythrocytes, and the serum was preincubated in PBS, 0.007 M Mg2ϩ, 0.01 M EGTA (pH 7.2) to make the hemolytic assay specific for the alternative pathway
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
