C3 Mouse
Description
Definition and Types of C3 Mouse Models
C3 Mouse models are genetically modified to study the role of C3 in health and disease. Common types include:
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C3 Knockout (KO) Mice: Lacking the C3 gene entirely, these mice exhibit deficiencies in complement activation and are prone to infections but show reduced inflammatory responses in certain contexts .
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Humanized C3 Mice: Genetically engineered to replace the mouse C3 gene with the human homolog, enabling studies of human-specific C3 interactions and diseases .
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Conditional KO Mice: Allow tissue-specific or age-specific deletion of C3 (e.g., in brain or immune cells) to dissect its role in development, aging, or neurodegeneration .
Biological Role of C3
C3 is central to all three complement pathways (classical, alternative, lectin) and mediates:
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Opsonization: C3b binds pathogens via a reactive thioester, marking them for phagocytosis .
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Inflammation: C3a is a potent anaphylatoxin, triggering immune responses and vascular permeability .
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Immune Regulation: C3 fragments (e.g., C3d) enhance B-cell responses, while iC3b modulates phagocytosis .
Research Applications of C3 Mouse Models
C3 Mouse models are invaluable for studying complement-related diseases and biological processes:
Kidney Diseases
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C3 Glomerulopathy (C3G): Humanized C3 mice develop severe glomerular C3 deposition, mesangial expansion, and rapid kidney failure, mimicking human C3G .
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C3 Deficiency Models: C3 KO mice show reduced kidney damage in some inflammatory contexts but increased susceptibility to infections .
Neurological Disorders
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Epilepsy and Cognitive Decline: C3 KO mice are protected against status epilepticus (SE)-induced recognition memory deficits, suggesting C3’s role in neuroinflammation .
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Key Findings:
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SE-induced memory deficits in WT mice are absent in C3 KO mice.
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Reduced astrocyte activation (GFAP levels) in C3 KO mice after SE.
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Alzheimer’s Disease (AD): Conditional C3 KO mice show preserved synapses and improved memory in aging and AD models .
Inflammatory Bowel Disease (IBD)
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C3 KO Mice: Exhibit enhanced inflammatory responses in the mid colon, with elevated NF-κB and ASC-inflammasome activity .
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Key Findings:
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Increased TNF, IL-6, IL-1α in C3 KO mice.
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Reduced tight junction proteins (E-cadherin, ZO-1) and ion channels.
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Key Research Findings
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C3 and Inflammation: C3 deficiency paradoxically exacerbates inflammation in some contexts (e.g., colitis) by dysregulating NF-κB and ASC pathways .
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C3 in Neurodegeneration: C3 KO mice resist SE-induced cognitive deficits and show reduced astrocyte activation, implicating C3 in seizure-related brain injury .
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Therapeutic Targets: Blocking C3b (anti-C3b mAbs) or C5 (eculizumab) improves outcomes in C3G and neuroinflammatory models .
Methodological Insights
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ELISA and Western Blot: Used to quantify C3 levels and activation products (e.g., C3b, iC3b) in serum and tissues .
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Immunohistochemistry: Identifies C3 deposition in glomeruli or brain regions (e.g., hippocampus) .
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Behavioral Assays: Novel object recognition (NOR) tests assess cognitive deficits in epilepsy models .
Product Specs
Complement component 3 (C3) is pivotal in activating all three complement pathways. Each pathway's initiation generates proteolytic enzyme complexes, binding to the target surface. These enzymes cleave C3, releasing the anaphylatoxin C3a and activating C3b. Notably, most activated C3 doesn't attach to the surface; its thioester reacts with water, forming fluid-phase C3b, quickly inactivated by factors H and I into iC3b. Surface-bound C3b is crucial across all pathways for effective C5 activation and the formation of C5b-9 complexes, ultimately lysing the target cell membrane.
Murine Complement Component 3 (C3) derived from mouse plasma, exhibiting a molecular weight of 185 kDa.
A solution subjected to sterile filtration.
The C3 solution is prepared in a phosphate-buffered saline (PBS).
Mouse C3 remains stable at 4°C for 2-4 weeks, provided the entire vial is used within this period. For extended storage, freeze at temperatures below -20°C. Adding a carrier protein like 0.1% HSA or BSA is recommended for long-term storage. Minimize freeze-thaw cycles.
Purity exceeds 93.0%, as determined by SDS-PAGE analysis.
Complement C3, C3 and PZP-like alpha-2-macroglobulin domain-containing protein 1, C3, CPAMD1.
Mouse Plasma.
Q&A
What is a C3 knockout (C3-/-) mouse?
C3 knockout mice are genetically modified models where the complement component 3 (C3) gene has been inactivated. These models are typically generated by replacing specific regions of the C3 gene, such as the 5'-flanking region and first 105 bp of exon 1, with a neomycin-resistance gene cassette . This genetic disruption results in mice that lack detectable C3 protein in serum and have no functional complement activity. The absence of this central complement component makes these mice valuable for studying the role of C3 in immune responses and disease processes.
How do researchers confirm C3 deficiency in knockout models?
Confirmation of C3 deficiency requires multiple analytical approaches:
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Serum analysis for C3 protein detection using immunological techniques
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Hemolytic complement activity assays to verify functional absence of the complement cascade
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Northern blot analysis to assess C3 mRNA expression in various tissues
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PCR-based genotyping to confirm targeted gene deletion
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ELISA-based quantification using specialized kits designed for mouse C3 detection
What phenotypic differences are observed between C3-deficient and wild-type mice?
C3-deficient mice exhibit several distinctive characteristics:
How do C3-deficient mice respond to bacterial challenges?
C3-deficient mice demonstrate dramatically increased susceptibility to bacterial infections, though responses vary across pathogens and disease stages. When challenged with Streptococcus pneumoniae, C3-/- mice exhibit approximately 2000-fold increase in bacteremia compared to littermate controls . In Chlamydia psittaci infection studies, C3-/- mice are approximately 100 times more susceptible than wild-type counterparts, with all C3-/- mice eventually succumbing to infection while the majority of wild-type mice recover .
What mechanisms explain the differential susceptibility of C3-deficient mice to pathogens?
The increased susceptibility of C3-deficient mice to bacterial infections results from multiple compromised immune mechanisms:
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Impaired opsonization of bacteria, reducing phagocytic clearance
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Diminished formation of the membrane attack complex
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Altered inflammatory signaling pathways
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Modified adaptive immune responses
Research suggests that in C. psittaci infection, the absence of C3 may initially limit harmful inflammatory responses (explaining early milder symptoms), but ultimately compromises pathogen clearance, allowing bacterial proliferation and overwhelming disease .
How reliable are antibody responses in C3-deficient mice during infection?
Despite C3's role as a B-cell co-activator through C3d, C3-/- mice can still generate substantial antibody responses to pathogens. In C. psittaci infection models, C3-/- mice produce anti-chlamydial IgM, total IgG, and IgG2a antibodies, though the IgM response appears delayed compared to wild-type mice . By day 21 post-infection in low-dose challenge experiments, C3-/- mice develop anti-chlamydial IgG2a levels that equal or exceed those in wild-type mice.
What control groups should be included in studies using C3-deficient mice?
Effective experimental design with C3-deficient mice requires appropriate controls to isolate C3-specific effects:
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Wild-type littermates (C3+/+) with identical genetic background
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Heterozygous mice (C3+/-) for dose-response evaluations
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Mock-infected or sham-treated controls for disease models
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Mice deficient in other complement components (e.g., C5-/- mice) to distinguish C3-specific effects from broader complement deficiencies
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Time-matched controls to account for the biphasic nature of some disease responses in C3-/- mice
How should researchers account for time-dependent effects in C3-deficient mouse studies?
The time-dependent effects observed in C3-deficient mice require specific experimental considerations:
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Design longitudinal studies with multiple timepoints to capture the complete disease progression
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Be aware that the most severely affected C3-/- mice may die before experiment endpoints, potentially skewing results toward less severe outcomes
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Consider that "the effect of the C3 gene knockout at later time-points was most likely even higher than determined by the analysis of the less affected survivors"
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Include both early (days 4-9) and late (days 9-21) assessment points to capture the biphasic response pattern
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Monitor immune parameters alongside clinical indicators to correlate physiological changes with disease progression
What are the optimal methods for quantifying C3 in mouse samples?
Accurate quantification of C3 in mouse samples requires specific methodological approaches:
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ELISA-based assays specifically designed for mouse C3 detection provide the most reliable quantitative results
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Four Parameter Logistic (4PL) curve fitting should be used for data analysis
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Standard curves plotting concentration versus absorbance on linear axes optimize accuracy
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Results should include %CV, Standard Deviation, and Standard Error calculations for quality control
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Samples requiring dilution must have appropriate dilution factors applied to final calculations
How does C3 expression vary across tissues in normal and knockout models?
C3 expression demonstrates significant tissue-specific patterns that have important implications for research:
In C3-deficient models, the absence of C3 mRNA in the liver contrasts with detectable C3 transcripts in other tissues. This results from transcription initiated in the neo cassette inserted during gene targeting, producing an altered C3 transcript that yields a primary translation product lacking the leader peptide .
What implications do extrahepatic C3 transcripts have for research using C3-deficient mice?
The presence of extrahepatic C3 transcription in C3-deficient mice has several important implications:
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While these mice lack functional secreted C3 protein, they may produce alternative C3 forms in specific tissues
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Metabolic labeling reveals synthesis of pro-C3 in lung tissue and peritoneal macrophages, but without post-synthetic processing or mature C3 secretion
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These mice serve as valuable models for studying tissue-specific C3 gene regulatory elements
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Researchers should assess both circulating and local C3 production when interpreting results
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The presence of modified C3 transcripts may influence tissue-specific immune responses in ways distinct from systemic C3 deficiency
How does C3 deficiency affect inflammatory lung conditions beyond infection?
C3 appears to play unexpected roles in pulmonary inflammation during autoimmune conditions. In an angiotensin II receptor type 1 (AT1R)-induced experimental systemic sclerosis model, C3-deficient mice developed more severe pulmonary inflammation than wild-type controls, despite similar anti-AT1R antibody levels . This contradicts the traditional view of complement as primarily pro-inflammatory.
C3-deficient mice also demonstrated increased rates of pulmonary cell apoptosis, with the apoptosis rate correlating with the degree of lung inflammation. These findings suggest C3 serves an anti-apoptotic and anti-inflammatory function in certain pulmonary autoimmune contexts .
How can researchers distinguish between different complement activation pathways using C3 mouse models?
C3 mouse models can help distinguish between different complement pathways through strategic experimental design:
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Compare C3-/- mice with mice deficient in specific complement receptors (CR1, CR2, CR3) to isolate receptor-mediated effects
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Use C3aR-deficient mice alongside C3-/- mice to distinguish C3a anaphylatoxin effects from other C3 functions
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Include C5-deficient mice to determine if phenotypes depend on terminal complement components downstream of C3
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Studies with C. psittaci have ruled out C5aR and membrane attack complex (MAC) as responsible for the increased susceptibility in C3-/- mice, suggesting C3a/C3aR and C3b/CRs as the critical mediators
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Utilize pathway-specific inhibitors alongside genetic models for validation
What are the current hypotheses explaining the divergent roles of C3 in early versus late infection stages?
Research suggests several hypotheses to explain C3's dual role during infection:
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Early pro-inflammatory role: Initial C3 activation may contribute to inflammation-associated tissue damage, explaining why C3-/- mice initially show milder symptoms in some models
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Late protective role: C3's opsonization and effector functions become critical for pathogen clearance in later infection stages
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Temporal regulation of cytokines: C3-/- mice show altered IFN-γ levels that don't simply correlate with bacterial load, suggesting complex regulatory mechanisms
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Differential engagement of complement receptors: Early vs. late infection may involve different complement receptors with opposing effects
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Compensatory mechanisms: Initial compensation for C3 deficiency may temporarily mask defects that become apparent later
Product Science Overview
Complement C3 is a large glycoprotein that is synthesized primarily in the liver and then secreted into the bloodstream. It is central to the activation of all three pathways of complement activation: the classical pathway, the lectin pathway, and the alternative pathway .
- Classical Pathway: Initiated by antibodies bound to antigens.
- Lectin Pathway: Triggered by mannose-binding lectin binding to pathogen surfaces.
- Alternative Pathway: Activated directly by pathogen surfaces.
Each pathway generates proteolytic enzyme complexes known as C3 convertases, which cleave C3 into two fragments: C3a and C3b .
- C3a: Functions as an anaphylatoxin, mediating inflammatory responses.
- C3b: Plays a key role in opsonization, tagging pathogens for destruction by phagocytes. It also participates in the formation of the C5 convertase, which is essential for the assembly of the membrane attack complex (MAC) that lyses target cells .
The activation of C3 is a pivotal step in the complement cascade. The cleavage of C3 and the subsequent binding of C3b to pathogen surfaces mark the pathogens for destruction. This process is known as opsonization and is crucial for the efficient clearance of pathogens by phagocytic cells such as macrophages and neutrophils .
Additionally, the breakdown products of C3b, such as iC3b and C3d, are recognized by various receptors on immune cells. These interactions enhance antigen presentation and stimulate the adaptive immune response, leading to the expansion of antigen-specific B-cell and T-cell populations .
Complement C3 is extensively studied in immunology and pathology. Researchers use various assays, such as ELISA (Enzyme-Linked Immunosorbent Assay), to measure the levels of C3 in mouse serum and plasma samples . These studies help in understanding the role of C3 in immune responses and its involvement in various diseases, including autoimmune disorders, infections, and inflammatory conditions.
