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• W2005961909 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 andD17H6S45, 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. 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 andD17H6S45, 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. The alternative pathway (AP) 1The abbreviations used are: AP, alternate pathway; ES, embryonic stem; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; HAGG, heat-aggregated murine IgG; RT, reverse transcriptase; bp, base pair(s); kb, kilobase pair(s). 1The abbreviations used are: AP, alternate pathway; ES, embryonic stem; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; HAGG, heat-aggregated murine IgG; RT, reverse transcriptase; bp, base pair(s); kb, kilobase pair(s). of complement activation forms an ancient part of the innate immune system (1Farries T.C. Atkinson J.P. Immunol. Today. 1991; 12: 295-299Abstract Full Text PDF PubMed Scopus (101) Google Scholar). The primary role of the AP probably lies in the immediate response to local infection by extracellular pathogens and can proceed on many microbial surfaces in the absence of specific antibody. The AP is also activated after C3b deposition by the classical pathway of complement activation and serves to amplify the classical pathway response by depositing more C3b on the surface of immune complexes. Factor B is a serine protease that, in conjunction with C3b activated by the serine protease factor D, forms the C3/C5-convertase of the AP. Activation by factor D cleaves factor B into Ba, a small peptide released into the fluid phase, and Bb, which contains the serine protease domain and remains associated with C3b in the C3/C5-convertase. The murine factor B gene (H2-Bf) is located in the S region of the major histocompatibility complex class III region (2Chaplin D.D. Woods D.E. Whitehead A.S. Goldberger G. Colten H.R. Seidman J.G. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 6947-6951Crossref PubMed Scopus (111) Google Scholar). The gene lies directly adjacent to the gene for complement component C2, the polyadenylation signal of C2overlapping with the 5′-regulatory region of H2-Bf (3Wu L.C. Morley B.J. Campbell R.D. Cell. 1987; 48: 331-342Abstract Full Text PDF PubMed Scopus (62) Google Scholar, 4Nonaka M. Gitlin J.D. Colten H.R. Mol. Cell. Biochem. 1989; 89: 1-14Crossref PubMed Scopus (19) Google Scholar). The 3′-untranslated region of H2-Bf overlaps with the 3′-untranslated region of the D17H6S45 gene (5Taylor P.R. Slingsby J.H. Walport M.J. Botto M. Mamm. Genome. 1997; 8: 796-797Crossref PubMed Scopus (1) Google Scholar), a gene with an unusual periodic structure and no defined function, formerly known as Rd (6Levi Strauss M. Carroll M.C. Steinmetz M. Meo T. Science. 1988; 240: 201-204Crossref PubMed Scopus (77) Google Scholar). Factor B is primarily synthesized in the liver (7Alper C.A. Raum D. Awdeh Z.L. Petersen B.H. Taylor P.D. Starzl T.E. Clin. Immunol. Immunopathol. 1980; 16: 84-89Crossref PubMed Scopus (92) Google Scholar) and has been shown to be produced by hepatocytes (8Morris K.M. Aden D.P. Knowles B.B. J. Clin. Invest. 1982; 70: 906-913Crossref PubMed Scopus (183) Google Scholar) and a wide variety of extrahepatic cells, including mononuclear phagocytes (9Whaley K. J. Exp. Med. 1980; 151: 501-516Crossref PubMed Scopus (242) Google Scholar,10Cole F.S. Schneeberger E.E. Lichtenberg N.A. Colten H.R. Immunology. 1982; 46: 429-441PubMed Google Scholar), fibroblasts (11Katz Y. Cole F.S. Strunk R.C. J. Exp. Med. 1988; 167: 1-14Crossref PubMed Scopus (52) Google Scholar), and epithelial and endothelial cells (12Strunk R.C. Eidlen D.M. Mason R.J. J. Clin. Invest. 1988; 81: 1419-1426Crossref PubMed Scopus (213) Google Scholar, 13Ripoche J. Mitchell J.A. Erdei A. Madin C. Moffatt B. Mokoena T. Gordon S. Sim R.B. J. Exp. Med. 1988; 168: 1917-1922Crossref PubMed Scopus (80) Google Scholar). As well as being an essential component of the AP C3/C5-convertase there has also been some evidence that the activation fragments of factor B may have roles as B cell growth factors (14Praz F. Ruuth E. J. Exp. Med. 1986; 163: 1349-1354Crossref PubMed Scopus (19) Google Scholar, 15Peters M.G. Ambrus Jr., J.L. Fauci A.S. Brown E.J. J. Exp. Med. 1988; 168: 1225-1235Crossref PubMed Scopus (43) Google Scholar), stimulants of mononuclear cell cytotoxicty (16Hall R.E. Blaese M. Davis III, A.E. Decker J.M. Tack B.F. Colten H.R. Muchmore A.V. J. Exp. Med. 1982; 156: 834-843Crossref PubMed Scopus (25) Google Scholar, 17Leijh P.C.J. van den Barselaar M. Daha M.R. van Furth R. J. Immunol. 1982; 129: 332-337PubMed Google Scholar), inducers of macrophage spreading (18Gotze O. Bianco C. Cohn Z.A. J. Exp. Med. 1979; 149: 372-386Crossref PubMed Scopus (58) Google Scholar), and immunosuppressants (19Ambrus Jr., J.L. Peters M.G. Fauci A.S. Brown E.J. J. Immunol. 1990; 144: 1549-1553PubMed Google Scholar). Deficiencies of components of the AP are extremely rare, and those that have been described presented in patients with very severe neisserial infections. The first factor B deficiency has only very recently been described in a 6-year-old boy who presented with a very severe meningococcal infection, an antigenically abnormal factor B protein, and no detectable factor B functional activity (20Densen P. Weiler J. Ackerman L. Barson B. Zhu Z.-B. Volanakis J. Mol. Immunol. 1996; 33 (abstr.): 68Google Scholar). In contrast, deficiencies of the classical pathway components are more common than those of the alternative pathway, and studies of humans with classical pathway component deficiencies have greatly increased our understanding of the roles of complement in vivo. Early classical pathway component deficiencies are strongly associated with immune complex-mediated diseases such as systemic lupus erythematosus. Complement-deficient humans (21Davies K.A. Erlendsson K. Beynon H.L.C. Peters A.M. Steinsson K. Valdimarsson H. Walport M.J. J. Immunol. 1993; 151: 3866-3873PubMed Google Scholar), and more recently a genetically engineered model of classical pathway deficiency, a C1q-deficient mouse (22Botto M. Nash J. Taylor P. Bygrave A. Pandolfi P.P. Loos M. Davies K.A. Walport M.J. Mol. Immunol. 1996; 33 (abstr.): 71PubMed Google Scholar), have both been demonstrated to have abnormal immune complex processing. To further our understanding of the physiological roles of the alternative pathway and factor B in vivo, we generated by homologous recombination in embryonic stem (ES) cells a mouse deficient in factor B by replacing exons 1 to 18 of the gene with a promoterlesslacZ reporter gene and the neomycin resistance gene.H2-Bf-deficient mice are fully viable when kept under specific pathogen-free conditions, and the disrupted allele was inherited in accordance with normal Mendelian patterns. The mice showed no gross phenotypic abnormalities; however, we found that the expression of the two neighboring genes, C2 andD17H6S45, was reduced and that the mice were functionally deficient in the classical as well as the alternative pathway. Mice with a disrupted H2-Bf locus were generated by homologous recombination in ES cells (23Thomas K.R. Capecchi M.R. Cell. 1987; 51: 503-512Abstract Full Text PDF PubMed Scopus (1790) Google Scholar) (Fig.1 A). The targeting vector pH2-Bf.lacZ/neo-HSVtk was constructed using isogenic DNA isolated from a 129/Sv genomic DNA λ library (Stratagene). All of the exons of H2-Bf from 1 to 18 (the last) corresponding to amino acids 4 onward and encompassing approximately 6 kb of genomic DNA were replaced with a 4.6-kb cassette consisting of a promoterlesslacZ reporter gene linked to a positively selectable marker, the neomycin resistance gene (neo) under the control of the pMC1 promoter. The lacZ/neo cassette was flanked on the 5′ side by a 4.6-kb region of genomic DNA from exon 9 of C2 to exon 1 of H2-Bf and on the 3′ side by a 2.8-kb genomic fragment from exon 18 of H2-Bf to exon 7 of theD17H6S45 gene. External to the 5′ region of homology with the target locus was placed a negatively selectable marker gene, the herpes simplex virus thymidine kinase gene (HSVtk). ES cells were transfected by electroporation with the linearized targeting vector pH2-Bf.lacZ/neo-HSVtk. Colonies surviving positive/negative selection (24Mansour S.L. Thomas K.R. Capecchi M.R. Nature. 1988; 336: 348-352Crossref PubMed Scopus (1308) Google Scholar) were isolated and screened by Southern blot (25Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) of EcoRI-digested genomic DNA using a 3′-external probe (P1) (Fig. 1 B). After identification of clones with a disrupted H2-Bf gene a 5′-external probe (P2) was used to confirm that genuine homologous recombination had occurred on both sides of the gene. A third probe within the targeting vector was used to confirm that the clones contained only the single copy of the targeting vector. Recombinant clones were microinjected into 3.5-day postcoitus blastocysts of C57BL/6 mice. The chimeric mice generated were bred with female C57BL/6 mice, and germline transmission was detected by the presence of agouti offspring, which were screened for the disrupted allele. All experimental animals were adults (between 8 and 12 weeks of age) and of a mixed genetic background, (129/Sv × C57BL/6) F2. Wild type (H2-Bf+/+) and heterozygous (H2-Bf+/−) littermates matched for sex and age were used as controls for H2-Bf-deficient (H2-Bf−/−) mice. For all in vitro hemolytic assays, male mice were used. When serum was required, blood samples were taken and left to coagulate on ice; the serum was used the same day and was kept on ice at all times. Total RNA was extracted from liver using guanidinium thiocyanate-CsCl density gradient centrifugation (25Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Total RNA was electrophoresed in 0.8% agarose 2.2 m formaldehyde gels and Northern blotted onto Hybond N+ (Amersham International, Buckinghamshire, UK) nylon membranes. First strand cDNA synthesis was undertaken using Superscript reverse transcriptase (Life Technologies, Inc.) as directed by the manufacturer. For amplification of the C2 cDNA the primers C2/10 (5′-CATCTATGCTATTGGGGTGG-3′) and C2/13b (5′-CCGAGATAGCTTCAGCAAGGC-3′) were used to generate a product of 492 bp. The 5′-untranslated region of H2-Bf cDNA was amplified with the primers Bf/5′ (5′-TGTAGCCAGATCCAGCATTTG-3′) and Bf/1b (5′-TCTCCATTGTCATGGAAAGC-3′), producing a 118-bp product.D17H6S45 cDNA was amplified with the primersRd/8+ (5′-GAACACACTGTATGTGTATGG-3′) and Rd/10− (5′-CAAGGGAGCCCCAGACAGATT-3′), yielding a 262-bp product. The cDNA from the gene for complement component C4 (C4) was amplified with the primers C4/2+ (5′-GAAGGACTTTAAGCTGAGCTC-3′) and C4/3− (5′-CTGAGCTACCAGCTGGATGTG-3′), resulting in a 129-bp product. Control primers for the amplification of theC1qA gene that generated a fragment of 339 bp were:C1qAI/1 (5′-TGGACAGTGGCTGAAGATGTC-3′) andC1qAII/1 (5′-ATAACCACGTTGCCAAGCGCT-3′). The cDNA ofC2, D17H6S45, C4, C1qA, andH2-Bf were amplified under the same conditions (1 unit of BIOTaq DNA polymerase (Bioline UK Ltd.), 2.5 mmMg2+, 0.2 mm dNTPs) using 20, 25, and 30 thermocycles, to give an estimation of the relative quantities of message present, each one being co-amplified with the C1qAgene to control for variability in the cDNA synthesis and PCR reactions. A previously describedin vitro assay with a trypsin-activated cobra venom factor-dependent convertase complex was used to detect functional factor B activity in mouse serum (26Paolucci E.S. Shreffler D.C. Immunogenetics. 1983; 17: 67-78Crossref PubMed Scopus (5) Google Scholar). In brief, 20 μl of 10% v/v mouse serum was incubated with 10 μl of 100 units/ml cobra venom factor (a kind gift from M. B. Pepys, Immunological Medicine, Imperial College School of Medicine), in the presence of 1 mm Mg2+, at 37 °C for 10 min and activated by incubation with 10 μl of 1 mg/ml bovine trypsin (Sigma-Aldrich, Poole, UK). The trypsin was inhibited with 10 μl of 2.5 mg/ml soybean trypsin inhibitor (Sigma-Aldrich), and the late components of complement were provided by the addition of 50 μl of 10 mm EDTA/normal human serum. Guinea pig erythrocytes (2 × 108 cells in 50 μl) were added and incubated at 37 °C for 90 min. The reaction was quenched by the addition of 1 ml of ice-cold PBS, and the amount of lysis was determined by the absorption at 413 nm of the supernatant after centrifugation at 10,000 × g for 1 min. 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 (27Andrews B.S. Theofilopoulos A.N. J. Immunol. Methods. 1978; 22: 273-281Crossref PubMed Scopus (24) Google Scholar). 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 Mg2+, 0.01m EGTA (pH 7.2) was used instead of sensitized sheep erythrocytes, and the serum was preincubated in PBS, 0.007m Mg2+, 0.01 m EGTA (pH 7.2) to make the hemolytic assay specific for the alternative pathway. Sheep erythrocytes were sensitized with a subagglutinating titer of rabbit hemolytic serum (TCS, Buckinghamshire, UK). 100 μl of cells at a concentration of 5 × 108 cells/ml were incubated for 15 min at 37 °C with 100 μl of mouse serum diluted 1:5 in PBS, 1% bovine serum albumin. The cells were pelleted by microfugation at 10,000 × g for 1 min, and the supernatant was removed. Fluid phase C3 was removed by repeated washing of the cells with ice-cold PBS, 1% bovine serum albumin. The cell pellet was resuspended in a final volume of 200 μl, and 50 μl were added of a 20 μg/ml suspension of a 125I-radiolabeled rat IgG2a monoclonal antibody against murine C3 (Connex, Martinsreid, Germany). The anti-C3 monoclonal antibody was radiolabeled by the direct IODO-GEN method (28Fraker P.J. Speck Jr., J.C. Biochem. Biophys. Res. Commun. 1978; 80: 849-857Crossref PubMed Scopus (3612) Google Scholar) to a specific activity of 18.5 MBq/mg. After incubation for 30 min at 37 °C with frequent agitation, two 75-μl aliquots of the cell suspension were removed and spun through 150 μl of di-“isononyl” phthalate oil (Fluka Chemicals, Gillingham, UK) at 10,000 ×g for 5 min. The tubes were cut, and the radioactivity of the cell pellet was used to determine the number of molecules of anti-C3 monoclonal present as a measure of C3 molecules bound per erythrocyte. Serum treated with EDTA to a concentration of 50 mm was used as a negative control, and serum treated with PBS, 0.007 m Mg2+, 0.01 m EGTA was used as a control for AP activity. Heat-aggregated murine IgG (HAGG) was used as a model immune complex. HAGG was prepared by incubation of total murine IgG (Sigma-Aldrich), at a concentration of 10 mg/ml, at 56 °C for 30 min, and insoluble complexes were removed by centrifugation at 1,100 × g for 10 min. The HAGG was then radiolabeled by the direct IODO-GEN method (28Fraker P.J. Speck Jr., J.C. Biochem. Biophys. Res. Commun. 1978; 80: 849-857Crossref PubMed Scopus (3612) Google Scholar) to a specific activity of 37 MBq/mg.H2-Bf-deficient animals and their wild type and heterozygous littermates were injected intravenously with 1 μg of125I-labeled HAGG (equivalent to 37 kBq of radioactivity). HAGG was injected with an equal volume of a 10% suspension of51Cr-labeled mouse erythrocytes as a blood pool tracer to control for injection quality. The mouse red blood cells were labeled by incubating 4 ml of a 10% suspension in PBS with 7.4 MBq of51Cr for 60 min at 37 °C. The cells were washed several times with PBS after labeling to remove unbound radioactivity. Two hours after the injection the mice were killed, a 50-μl blood sample was taken, and the spleen and liver were removed and weighed.125I and 51Cr radioactivity was determined using a γ-scintillation counter and corrected for emission window overlap. The unbound radioactivity present in the intravascular compartment of the spleen was determined from the published murine splenic blood volume of 180 μl/g (29Altman P.L. Dittmer D.S. Blood and Other Bodily Fluids. Biological Handbooks, Federation of American Societies for Experimental Biology, Bethesda, MD1961: 9Google Scholar), and the 125I-HAGG was measured in a unit blood volume. Immune complex uptake was defined as: (125I cpm of the whole organ − 125I cpm in blood pool of the organ)/organ mass. Analysis of the51Cr radioactivity in the blood sample confirmed consistency in injection quality. All methods used to calculate the organ distribution of the HAGG, including percentage of injected HAGG in the organ, counts/min/g of organ mass and counts/min of the whole organ gave consistent results, whether corrections were made for blood pooling effects or not. The murine H2-Bf gene was disrupted in ES cells by homologous recombination with the targeting vector pH2-Bf.lacZ/neo-HSVtk. A targeting frequency of approximately 1 in 16 of the double selection resistant ES cell clones was achieved at the H2-Bf locus. Five of the recombinant clones were chosen, determined to have a euploid karyotype, and microinjected into 3.5-day postcoitus blastocysts of C57BL/6 mice. Four of the five clones generated chimeras that transmitted the disrupted allele through the germline. Four independent lines ofH2-Bf-deficient animals (Bf-16, Bf-40,Bf-79, and Bf-114) were established in both the mixed (129/Sv × C57BL/6) and pure (129/Sv) genetic backgrounds.H2-Bf-deficient mice had neither detectable factor B mRNA by Northern blot (Fig. 2), using a cDNA probe that encodes the Ba activation fragment, nor protein in their serum by radial-immunodiffusion using a cross-reacting rabbit anti-human factor B polyclonal antisera (Calbiochem, Nottingham, UK).H2-Bf-deficient animals showed no specific functional factor B activity in the cobra venom factor-dependent assay (data not shown) and no detectable alternative pathway activity as demonstrated by their failure to lyse guinea pig erythrocytes (data not shown). The mice were fully viable under specific pathogen-free conditions, and the disrupted allele was inherited in accordance with normal Mendelian inheritance patterns (the genotypes of 296 offspring from F1 heterozygous crosses were 77 H2-Bf−/− (26%), 146 H2-Bf+/− (49.3%), and 73 H2-Bf+/+ (24.7%)). Northern blot analysis of the neighboring gene for complement component C2 failed to detect C2 mRNA in liver RNA ofH2-Bf-deficient mice (Fig. 2), while the transcript was visible in the wild type mice. Overexposure of the same film showed a very weak signal of the correct size in the gene-targeted mice (data not shown). To better quantify theC2 mRNA present, a semiquantitative RT-PCR was used.C2 cDNA was amplified at the same time as cDNA for the C1qA gene using 20, 25, and 30 cycles. C2product was not detectable after 20 amplification cycles in the deficient mice, whereas it was already visible in the wild type littermates (Fig. 3). After 25 cycles, the message was detected in the deficient mice, but the product was much more abundant in the wild type mice. Amplification of theC1qA gene was equivalent at all steps between all mice. This RT-PCR observation is consistent with the presence of C2mRNA, albeit at greatly reduced levels. The 5′-untranslated region of the H2-Bf gene was amplified in the same way, since this region of the mRNA should be retained in the deficient mice where only the H2-Bf coding sequence was replaced by thelacZ reporter gene. The results obtained by RT-PCR were very similar to those for C2 (Fig. 3). The 5′-untranslated region was not visible after 20 cycles of amplification in the deficient animals, whereas it could be seen in the wild type mice. After 25 cycles, the product was detectable from the deficient mice but again at much reduced levels compared with the wild type. Amplification of theC1qA cDNA was equal between deficient and sufficient mice. This latter observation suggests that the reporter gene was expressed at much lower levels than the wild type H2-Bfgene. This observation could not be confirmed by direct comparison on Northern blot as the 5′-untranslated region of H2-Bf did not work successfully as a probe. A similar pattern of gene expression was observed for the downstream neighbor gene of H2-Bf,D17H6S45 (Fig. 3). Expression of the gene for complement component C4 (C4) was checked to determine if the effect of the gene targeting was localized to these three genes or if it had spread further, since C4 is approximately 40 kb downstream of H2-Bf. The expression of C4 was found to be similar in H2-Bf-deficient and -sufficient mice (Fig. 3), suggesting that the effects of the gene-targeting event were localized. In the absence of an antigenic assay for mouse C2 we assessed the classical pathway-mediated total hemolytic complement activity in the serum. The percentage of total lysis (±S.E.) caused by serum fromH2-Bf+/+ (n = 5) and H2-Bf+/− (n = 5) mice was 49.3% (±8.13%) and 45.8% (±10.24%), respectively. The H2-Bf−/− (n= 5) mice were unable to lyse antibody-sensitized sheep erythrocytes (0.85 ± 0.37%). As a further test of the classical pathway activity, we measured the ability of the serum from the deficient animals to opsonize antibody-sensitized sheep erythrocytes with C3 and found that this was also absent (Fig. 4) and no different to the opsonizing capabilities of serum from C1q-deficient mice, which are specifically deficient of the classical pathway of complement activation. Serum from normal mice was able to bind nearly 1700 molecules of C3 per erythrocyte. The alternative pathway alone was unable to opsonize the antibody-sensitized erythrocytes, demonstrating the specific requirement for the classical pathway. To further confirm the total absence of classical pathway activity in these mice, we decided to evaluate immune complex processing in vivo. Immune complex processing has previously been shown to be abnormal in humans with classical pathway deficiency (21Davies K.A. Erlendsson K. Beynon H.L.C. Peters A.M. Steinsson K. Valdimarsson H. Walport M.J. J. Immunol. 1993; 151: 3866-3873PubMed Google Scholar) and also in C1q-deficient mice (22Botto M. Nash J. Taylor P. Bygrave A. Pandolfi P.P. Loos M. Davies K.A. Walport M.J. Mol. Immunol. 1996; 33 (abstr.): 71PubMed Google Scholar). Splenic uptake of radiolabeled heat-aggregated murine IgG was severely impaired in theH2-Bf-deficient mice (Fig.5 A). The difference in splenic uptake was analyzed using the Mann-Whitney U test. Two hours postinjection the median splenic uptake of HAGG was 23,180 cpm/g (range 12,380–30,350) in 10 gene-targeted mice, compared with 42,040 cpm/g (range 31,940–103,300) in 13 control mice (U = 0,p < 0.0001). Hepatic uptake in theH2-Bf-deficient mice was no different from the controls (Fig. 5 B). The median hepatic uptake in the 10H2-Bf-deficient mice was 35,460 cpm/g (range 20,510–49,390), compared with 27,680 cpm/g (range 10,450–64,320) in the 13 control mice (U = 53, p < 0.48). The organ distribution of the aggregated IgG was no different form that observed in C1q-deficient mice (data not shown). Splenic uptake of monomeric murine IgG was approximately 40% of the uptake of HAGG in the complement-deficient animals (data not shown), implying that very little splenic uptake of aggregates was occurring in the deficient animals. These results suggest an effective total failure of classical pathway activity in vivo.Figure 5Immune complex processing inH2-Bf- deficient mice. A, graph shows the counts/min/g of 125I-HAGG present in the spleen ofH2-Bf−/− mice and their H2-Bf+/+ litter mates 2 h postinjection. The values shown here have been corrected for radioactivity in the blood pool of the spleen using the previously reported formula (29Altman P.L. Dittmer D.S. Blood and Other Bodily Fluids. Biological Handbooks, Federation of American Societies for Experimental Biology, Bethesda, MD1961: 9Google Scholar). Each circle represents a single mouse, and the graph shows the results obtained from two separate experiments (closed and open circles) performed with two different lines of H2-Bf-deficient mice. Horizontal bars denote the medians of both groups. B, graph shows the counts/min/g of 125I-HAGG present in the liver of ofH2-Bf−/− mice and their H2-Bf+/+ littermates 2 h postinjection. The values shown here have been corrected as described above. The data shown is for the same mice depicted inA. Horizontal bars denote the medians of both groups.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Here we report a characterized total deficiency of complement component factor B. Mice were generated by homologous recombination in ES cells that carried a disrupted H2-Bf allele with the entire coding region of the wild type gene replaced with a promoterlesslacZ reporter gene cloned into the same reading frame as the first codons of H2-Bf. Downstream of the lacZgene was the positively selectable marker gene for neomycin resistance. Four independent ES cell clones generated lines of mice carrying the disrupted gene. Homozygous H2-Bf-deficient animals had no detectable levels of factor B in their serum by radial immunodiffusion and had no specific functional factor B activity in a cobra venom factor-dependent assay. H2-Bf-deficient animals had no AP activity demonstrated by their failure to lyse guinea pig erythrocytes. The mice were fully viable under specific pathogen-free conditions. In all four different lines of H2-Bf-deficient mice, transmission of the targeted allele was in accordance with normal Mendelian inheritance. This observation is not entirely in agreement with that of Matsumoto et al. (30Matsumoto M. Fukuda W. Circolo A. Goellner J. Strauss-Schoenberger J. Wang X. Fujita S. Hidvegi T. Chaplin D.D. Colten H.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8720-8725Crossref PubMed Scopus (173) Google Scholar), who have recently generated H2-Bf-deficient mice and found a selective disadvantage for the inheritance of their disrupted factor B allele in only one of their two mouse lines. This abnormal inheritance pattern may have been due to chance. Surprisingly, the mutation introduced into the H2-Bf gene caused a dramatic down-regulation in the expression of the neighboring gene for complement component C2 of the classical pathway. The regulatory elements of C2 expression reported in the literature are 5′ of C2, away from the site of the deletion (31Sullivan K.E. Wu L.C. Campbell R.D. Valle D. Winkelstein J.A. Eur. J. Immunol. 1994; 24: 393-400Crossref PubMed Scopus (5) Google Scholar), and at the 3′ end of the gene in the intragenic region betweenH2-Bf and C2 (32Moreira A. Wollerton M. Monks J. Proudfoot N.J. EMBO J. 1995; 14: 3809-3819Crossref PubMed Scopus (71) Google Scholar, 33Ashfield R. Patel A.J. Bossone S.A. Brown H. Campbell R.D. Marcu K.B. Proudfoot N.J. EMBO J. 1994; 13: 5656-5667Crossref PubMed Scopus (98) Google Scholar, 34Ashfield R. Enriquez Harris P. Proudfoot N.J. EMBO J. 1991; 10: 4197-4207Crossref PubMed Scopus (56) Google Scholar) unaffected by the targeting event. Expression of a second neighboring gene, D17H6S45, was similarly affected by the targeting event. There are four possible explanations for the observed phenomenon. (i) Regulatory elements forC2, D17H6S45," @default.
• W2005961909 created "2016-06-24" @default.
• W2005961909 creator A5027333125 @default.
• W2005961909 creator A5030200734 @default.
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• W2005961909 creator A5072004270 @default.
• W2005961909 date "1998-01-01" @default.
• W2005961909 modified "2023-10-11" @default.
• W2005961909 title "A Targeted Disruption of the Murine Complement Factor B Gene Resulting in Loss of Expression of Three Genes in Close Proximity, Factor B, C2, and D17H6S45" @default.
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