IFNG Mouse, His

What is mouse IFN-γ and how does it structurally differ from human IFN-γ?

Mouse IFN-γ (mIFN-γ) is a pleiotropic cytokine that functions as a macrophage-activating factor. It shares approximately 41% sequence identity with human IFN-γ (hIFN-γ) . Despite this relatively low homology, both variants perform similar immunoregulatory functions in their respective species. The active form of mouse IFN-γ exists as an antiparallel dimer that triggers the IFN-γ/JAK/STAT pathway . This dimerization is essential for receptor binding and subsequent biological activity.

The molecular weight of mouse IFN-γ is approximately 20 kDa, which may vary slightly when produced with a His-tag for purification purposes . Understanding these structural differences is crucial when designing cross-species experiments or interpreting results from mouse models in the context of human disease.

What are the primary cellular sources of mouse IFN-γ in vivo?

Mouse IFN-γ is produced by multiple immune cell populations including:

• Activated T lymphocytes (both CD4+ and CD8+ subsets)

• Natural killer (NK) cells

• B cells

Interestingly, research has demonstrated that mouse peritoneal macrophages can constitutively express IFN-γ, suggesting an autocrine regulatory mechanism that may play an important role in macrophage function and immune response coordination . This finding challenges the traditional view that IFN-γ production is restricted to lymphoid cells and highlights the complex network of cytokine regulation in the immune system.

How do post-translational modifications affect mouse IFN-γ activity?

Research indicates that glycosylation of mouse IFN-γ does not affect its biological activity . This finding has significant implications for recombinant protein production, as it suggests that non-glycosylated recombinant IFN-γ (such as that produced in bacterial expression systems like E. coli) should maintain full biological functionality.

For His-tagged mouse IFN-γ specifically, the addition of the histidine tag generally does not interfere with the protein's biological activity. This makes His-tagged versions particularly useful for research applications as they combine ease of purification with preserved functionality.

What are the primary signaling pathways activated by mouse IFN-γ?

Mouse IFN-γ initiates signaling by binding to its heterodimeric receptor composed of IFNGR1 and IFNGR2 subunits. This binding event triggers a signaling cascade primarily involving:

• Janus kinase 1 (JAK1)

• Janus kinase 2 (JAK2)

• Signal transducer and activator of transcription 1 (STAT1)

Upon receptor engagement, conformational changes in the receptor's intracellular domains activate JAK1 and JAK2, which subsequently phosphorylate STAT1. Phosphorylated STAT1 forms homodimers that translocate to the nucleus and bind to gamma-activated sequence (GAS) elements in the promoters of IFN-γ-responsive genes, inducing transcriptional changes that mediate IFN-γ's biological effects.

Can mouse IFN-γ regulate its own expression?

Yes, mouse IFN-γ can upregulate its own gene expression through an autocrine mechanism, particularly in peritoneal macrophages . When mouse peritoneal macrophages are treated with IFN-γ, a significant accumulation of IFN-γ mRNA occurs, followed by secretion of IFN-γ protein after 24-48 hours.

This self-regulatory capacity is not observed in mouse lymphocytes from mesenteric lymph nodes or in various mouse cell lines, indicating cell type specificity . The upregulation of IFN-γ expression can also be found in peritoneal macrophages from anti-asialo GM1-treated nude mice, further supporting an autocrine regulatory mechanism in these cells. This self-amplification loop may be critical for sustaining appropriate immune responses in specific tissue microenvironments.

What are the main biological functions of mouse IFN-γ in immune regulation?

Mouse IFN-γ orchestrates diverse biological functions related to host defense and immune regulation, including:

• Antiviral and antiparasitic defense mechanisms

• Apoptosis induction in target cells

• Inflammatory response regulation

• Innate and acquired immunity coordination

• Macrophage activation and polarization

• Cell proliferation inhibition (for both normal and transformed cells)

IFN-γ signaling recruits and activates various immune cell populations, including macrophages, natural killer cells, and cytotoxic T lymphocytes . This cytokine network creates a coordinated immune response against pathogens and tumors. Paradoxically, IFN-γ has also been implicated in resistance to NK cell and CTL responses and in immune escape mechanisms in certain cancer contexts .

What are the optimal conditions for reconstituting and storing His-tagged mouse IFN-γ?

For maximum stability and activity of His-tagged mouse IFN-γ, follow these guidelines:

Reconstitution Protocol:

• Lyophilized protein should be reconstituted in sterile water or appropriate buffer (typically PBS with 0.1% BSA as a carrier protein)

• Gently mix by swirling or pipetting; avoid vigorous vortexing that may cause protein denaturation

• Allow complete dissolution before determining concentration

Storage Recommendations:

• Long-term storage: -80°C in single-use aliquots to prevent freeze-thaw cycles

• Medium-term storage: -20°C for up to 3 months

• Working solution: 2-8°C for up to 1 week

Stability Considerations:

• Avoid repeated freeze-thaw cycles as they significantly compromise protein activity

• For His-tagged preparations specifically, monitor for potential aggregation as His-tags can sometimes promote protein-protein interactions

How should I design dose-response experiments with recombinant mouse IFN-γ?

When designing dose-response experiments with recombinant mouse IFN-γ, consider these methodological recommendations:

Concentration Range:

• For cell activation studies: 1-100 ng/mL, with typical effective concentrations around 5-20 ng/mL

• For ELISA standard curves: serial dilutions starting from 2000 pg/mL as recommended for commercial kits

Experimental Timeline:

• Acute responses (signaling activation): 15 minutes - 6 hours

• Gene expression changes: 6-24 hours

• Phenotypic alterations: 24-72 hours

Control Conditions:

• Include appropriate vehicle controls matching the reconstitution buffer

• Consider including a heat-inactivated IFN-γ control to confirm specificity

• For His-tagged proteins, compare with non-tagged versions in critical experiments to rule out tag-mediated effects

Readout Selection:

• Select appropriate readouts based on your research question (e.g., STAT1 phosphorylation for signaling, specific gene expression for transcriptional responses, functional assays for biological effects)

What methods are available for detecting and quantifying mouse IFN-γ in experimental samples?

Several complementary approaches can be used to measure mouse IFN-γ:

Enzyme-Linked Immunosorbent Assay (ELISA):

• Commercial kits available with detection ranges typically from 15-2000 pg/mL

• Suitable for cell culture supernatants, serum, plasma, and tissue homogenates

• Follow manufacturer protocols for sample dilution and preparation

Western Blotting:

• Detects mouse IFN-γ protein at approximately 17-20 kDa

• Useful for cell and tissue lysates

• Can confirm protein integrity and detect potential degradation products

Flow Cytometry:

• Intracellular staining for IFN-γ in specific cell populations

• Requires cell permeabilization and fluorescently-labeled antibodies

• Provides information about the cellular sources of IFN-γ in heterogeneous samples

Quantitative PCR (qPCR):

• Measures IFN-γ mRNA expression levels

• Requires appropriate reference genes for accurate normalization

• Useful for studying transcriptional regulation when protein detection is challenging

What are the key considerations when using His-tagged mouse IFN-γ in immunological assays?

When working with His-tagged mouse IFN-γ, researchers should consider these important factors:

Purification Advantages:

• The His-tag facilitates protein purification using nickel affinity chromatography

• Results in high-purity preparations suitable for sensitive immunological assays

• Allows for stringent washing conditions to remove contaminants

Potential Interference Considerations:

• While His-tags generally have minimal impact on protein function, they might:

  • Slightly alter protein folding or dimerization kinetics

  • Create non-specific interactions in certain experimental systems

  • Influence protein half-life in vivo

Validation Approaches:

• Compare biological activity with non-tagged versions in key experiments

• Consider tag removal using protease cleavage if a cleavage site is incorporated

• Include appropriate controls for non-specific effects of the tag itself

Detection Advantages:

• His-tagged preparations can be detected using anti-His antibodies, providing an additional detection method independent of IFN-γ-specific antibodies

• Useful for tracking protein distribution or confirming expression in complex systems

What roles does IFN-γ play in neuroinflammatory disease models?

Mouse IFN-γ has significant functions in neuroinflammatory disease contexts, as demonstrated in models of multiple system atrophy (MSA):

In a mouse model of MSA characterized by alpha-synuclein overexpression in oligodendrocytes, researchers observed:

• Infiltration of CD4+ and CD8+ T cells into the brain

• Increased CD4+ T-cells positive for the transcription factor T-bet

• Significant production of IFN-γ by infiltrating immune cells

Further mechanistic studies using genetic and pharmacological approaches demonstrated that:

• IFN-γ is produced primarily by infiltrating CD4+ T-cells

• IFN-γ mediates key pathological mechanisms driving MSA progression

• IFN-γ-neutralizing antibody treatment attenuated disease progression

These findings suggest IFN-γ represents a potential therapeutic target in MSA, with researchers noting that "IFNγ represents a potential future disease-modifying therapeutic target in multiple system atrophy" . This research highlights the importance of understanding IFN-γ's functions in specific disease contexts to develop targeted therapeutic approaches.

How should researchers design experiments to target IFN-γ signaling in mouse models?

When designing experiments to target IFN-γ signaling in mouse models, consider these methodological approaches:

Neutralizing Antibodies:

• Anti-IFN-γ monoclonal antibodies can effectively block IFN-γ activity in vivo

• Timing is critical - treatment before disease onset tests preventive effects, while treatment after onset assesses therapeutic potential

• Dosing frequency must account for antibody half-life and tissue distribution

• Researchers have successfully used IFN-γ-neutralizing antibody treatment in mouse models of MSA both before and after disease onset

Genetic Approaches:

• Complete IFN-γ knockout mice provide information about developmental and systemic effects

• Conditional knockout systems using Cre-lox technology allow for tissue-specific or temporal control of gene deletion

• CRISPR/Cas9-mediated editing enables precise modification of IFN-γ pathway components

Receptor Targeting:

• Blocking antibodies against IFNGR1 or IFNGR2 can inhibit signaling

• Small molecule inhibitors of downstream signaling components (JAK/STAT) provide alternative approaches

• Soluble receptor domains can act as decoys to sequester IFN-γ

Readout Selection:

• Include multiple readouts spanning signaling (STAT1 phosphorylation), transcriptional (IFN-γ-responsive genes), and functional (disease parameters) endpoints

• Account for potential compensatory mechanisms in chronic models

How do microbiome-host interactions influence IFN-γ responses in mouse models?

The interaction between the microbiome and IFN-γ responses in mouse models represents an emerging research area with important implications:

Airway Microbiota Interactions:

• Respiratory exposure to specific microbes can modulate airway inflammation and immune responses

• Studies incorporating "wild" mouse microbes in laboratory settings better align mouse models with human disease responses

• Natural members of the mouse microbiota, such as Bordetella pseudohinzii, can persist in the respiratory tract for months and influence immune parameters without causing overt illness

Experimental Approaches:

• Gnotobiotic versus conventional mice comparisons reveal microbiome-dependent differences in IFN-γ production

• Colonization with defined microbial communities allows for controlled studies of specific microbe-immune interactions

• Antibiotic treatment can temporarily alter microbiota composition to study acute effects on IFN-γ responses

Observed Effects:

• Airway colonization with B. pseudohinzii increased both effector T cells and T regulatory cells in mouse lungs

• Interestingly, this colonization preferentially increased IL-17A-expressing T cells rather than IFN-γ-producing Th1 cells

• This demonstrates how specific members of the microbiome can shape the balance between different T cell subsets and their cytokine production profiles

These findings highlight the importance of considering microbiome variables when studying IFN-γ responses in mouse models, particularly when translating results to human disease contexts.

What computational tools are available for predicting IFN-γ-inducing peptides in mouse models?

Researchers now have access to sophisticated computational tools for predicting IFN-γ-inducing peptides:

IFNepitope2:

• A host-specific technique for annotating IFN-γ inducing peptides

• Updated version of the IFNepitope platform introduced by Dhanda et al.

• Utilizes a dataset containing 7,983 experimentally validated IFN-γ inducing peptides specifically for mouse hosts

Methodological Approach:

• Combines machine learning techniques with similarity-based methods (BLAST)

• Employs compositional features, particularly dipeptide composition, which outperforms one-hot encoding or binary profile approaches

• The hybrid model achieved an AUROC of 0.85 for mouse host predictions

Applications:

• Design of peptide-based vaccines with enhanced immunogenicity

• Development of immunomodulatory peptides for therapeutic applications

• Screening potential epitopes from pathogen proteins for vaccine development

• Rational design of experimental tools to study IFN-γ responses in specific contexts

This resource is available as both a web server (https://webs.iiitd.edu.in/raghava/ifnepitope2/) and as a standalone application or Python package (https://github.com/raghavagps/ifnepitope2)[6].

How can mouse IFN-γ be used in cancer immunotherapy research?

Mouse IFN-γ serves as a valuable tool in cancer immunotherapy research due to its pleiotropic effects on immune responses and tumor cells:

Direct Anti-tumor Effects:

• Upregulates MHC class I expression on tumor cells, enhancing their recognition by cytotoxic T lymphocytes

• Induces growth arrest and apoptosis in certain tumor cell lines

• Inhibits angiogenesis, limiting tumor vascularization and growth

Immune Modulation Applications:

• Enhances dendritic cell maturation and antigen presentation

• Polarizes macrophages toward an M1 (anti-tumor) phenotype

• Augments NK cell cytotoxicity against tumor targets

Paradoxical Effects:

• IFN-γ is also implicated in resistance to NK cell and CTL responses

• Can contribute to immune escape mechanisms in various cancer types

• May induce PDL1 expression on tumor cells, potentially limiting anti-tumor immunity

Experimental Approaches:

• Combination with checkpoint inhibitors to overcome potential immunosuppressive effects

• Local delivery systems to achieve high intratumoral concentrations while limiting systemic toxicity

• Genetic engineering of tumor-infiltrating lymphocytes or CAR-T cells to produce IFN-γ at tumor sites

What are the latest techniques for studying IFN-γ-dependent pathways in mouse models?

Several cutting-edge approaches have emerged for studying IFN-γ-dependent pathways in mouse models:

Reporter Systems:

• IFN-γ reporter mice expressing fluorescent proteins under the control of the IFN-γ promoter

• Allow real-time visualization of IFN-γ expression patterns in vivo

• Can be combined with intravital microscopy for longitudinal studies

Single-cell Technologies:

• Single-cell RNA sequencing reveals cell-specific responses to IFN-γ stimulation

• Mass cytometry (CyTOF) enables simultaneous detection of multiple signaling nodes

• Spatial transcriptomics provides information about IFN-γ pathway activation in tissue context

CRISPR/Cas9 Applications:

• Precise gene editing to create novel mouse models with specific mutations in IFN-γ pathway components

• CRISPR screening approaches to identify new regulators of IFN-γ responses

• In vivo CRISPR delivery for tissue-specific modification of pathway components

Optogenetic and Chemogenetic Tools:

• Allow temporal and spatial control of IFN-γ signaling

• Enable studies of the acute versus chronic effects of pathway activation

• Provide unprecedented precision in manipulating specific aspects of the signaling cascade

Comparative Table: Mouse vs. Human IFN-γ Properties

Experimental Applications of Recombinant Mouse IFN-γ