IL3RA Mouse

What is IL3RA in mice and how does it differ from human IL3RA?

Mouse IL3RA (also known as CD123 or SUT-1) is a transmembrane protein that functions as the alpha subunit of the interleukin-3 receptor. It has a molecular weight of approximately 70-80 kDa and spans from Ser17 to Lys331 in its mature form . The mouse IL3RA system differs significantly from the human system in that mice possess two distinct IL-3 receptor beta proteins. The first identified mouse IL-3R beta was called AIC2A (now Csf2rb2), which is IL-3 specific. The second, AIC2B (now Csf2rb), doesn't bind IL-3 independently and is the homolog of the human IL-3R beta. This duplication event creates unique research considerations when translating findings between species .

What is the biological function of IL3RA in mice?

IL3RA functions as a cell surface receptor for IL-3 and is expressed on hematopoietic progenitor cells. When IL-3 binds to IL3RA, it induces heterodimerization with either of the beta subunits (Csf2rb or Csf2rb2) . This interaction triggers intracellular signaling cascades, including the JAK2/STAT5 pathway and PI3K activation, which control the proliferation and differentiation of hematopoietic progenitor cells into lineage-restricted cells . The IL3RA/IL-3 signaling axis is crucial for normal hematopoiesis and immune system development in mice, regulating the production of various white blood cell populations including granulocytes and monocytes-macrophages .

How can I detect and measure IL3RA expression in mouse samples?

For detecting mouse IL3RA, fluorochrome-conjugated antibodies designed for flow cytometry are the most common approach. Commercial antibodies like Mouse IL-3R alpha/CD123 Alexa Fluor® 488-conjugated Antibody are available and validated for direct ELISAs and Western blots . For flow cytometry experiments, optimal antibody dilutions should be determined for each specific application.

Methodological approach:

• Isolate cells from mouse tissue (bone marrow, spleen, or peripheral blood)

• Stain with fluorochrome-conjugated anti-IL3RA antibodies (e.g., Alexa Fluor 488-conjugated)

• Include appropriate isotype controls at the same concentration as the primary antibody

• Analyze using flow cytometry, gating on relevant cell populations

• For Western blot detection, prepare cell lysates and use validated anti-IL3RA antibodies

How should I design experiments to study IL3RA signaling in mouse models?

When designing experiments to investigate IL3RA signaling in mice, consider a multi-faceted approach:

• Receptor expression analysis: Quantify IL3RA and beta subunit (Csf2rb and Csf2rb2) expression ratios at both transcript and protein levels. This is critical as the IL3Rα/βc ratio affects receptor assembly and signaling outcomes .

• Signaling pathway investigation: Focus on JAK2/STAT5 activation, which is a primary downstream pathway of IL3RA signaling. Phosphorylation of these proteins can be measured using phospho-specific antibodies in Western blots or flow cytometry .

• Functional assays: Assess colony formation, proliferation, and differentiation of hematopoietic progenitors in response to IL-3 stimulation.

• Controls: Include:

  • Receptor knockout or knockdown controls

  • Pathway inhibitor controls (JAK2 inhibitors)

  • Comparison between wild-type and mutant receptors

  • Both IL3-stimulated and unstimulated conditions

What are the key considerations when using antibodies to study mouse IL3RA?

When selecting antibodies for mouse IL3RA research:

• Specificity verification: Confirm the antibody specifically recognizes mouse IL3RA with no cross-reactivity to human IL3RA or other proteins. For example, the Mouse IL-3R alpha/CD123 Alexa Fluor® 488-conjugated Antibody shows no cross-reactivity with recombinant human IL-3R in direct ELISAs and Western blots .

• Application compatibility: Verify the antibody is validated for your specific application (flow cytometry, Western blot, immunoprecipitation, etc.).

• Clone selection: Different monoclonal antibodies recognize different epitopes, which may be differentially accessible depending on receptor conformation or complex formation.

• Titration: Always optimize antibody concentration for your specific experimental system to minimize background and maximize signal-to-noise ratio .

• Controls: Include isotype controls at identical concentrations to the test antibody to account for non-specific binding .

How does IL3RA form different receptor complexes in mice, and what are the functional implications?

Mouse IL3RA forms two distinct classes of high-affinity IL3 receptors with different beta subunits:

• IL3RA + Csf2rb2 (AIC2A) complex: This complex is specific to IL-3 signaling and forms when IL3RA binds to the IL3-specific beta subunit Csf2rb2 .

• IL3RA + Csf2rb (AIC2B) complex: This complex utilizes the beta subunit that is also shared by high-affinity IL-5 and GM-CSF receptors .

These different complexes can form distinct higher-order structures including hexamers and dodecamers, as has been demonstrated in human systems . The ratio of IL3RA to beta subunits appears to influence the stoichiometry of these complexes, with higher IL3RA/βc ratios favoring hexamer formation while lower ratios may lead to dodecamer formation .

Functional implications:

• Different signaling intensity or duration depending on the complex formed

• Potential for cross-talk between IL-3, IL-5, and GM-CSF signaling pathways when the shared beta subunit is involved

• Altered downstream pathway activation that may influence cell fate decisions in hematopoietic progenitors

What signaling pathways are activated downstream of IL3RA in mice?

IL3RA signaling in mice activates multiple downstream pathways:

• JAK2/STAT5 pathway: Upon IL-3 binding, IL3RA heterodimerizes with its beta subunit, leading to phosphorylation and activation of JAK2, which subsequently phosphorylates STAT5. Activated STAT5 translocates to the nucleus and initiates a transcriptional program promoting cell proliferation and survival .

• PI3K pathway: IL3RA activation also leads to phosphorylation and activation of PI3K, which generates phosphatidylinositol-3,4,5-trisphosphate (PIP3), activating AKT and downstream effectors that regulate metabolism, survival, and proliferation .

• Ras/MAPK pathway: IL3RA signaling can activate the Ras proteins (Kras, Nras, Hras) leading to MAPK cascade activation, which regulates gene expression, cell growth, and differentiation .

These pathways work in concert to mediate the biological effects of IL-3 on hematopoietic cell proliferation, differentiation, and survival. The specific contribution of each pathway may vary depending on cell type and context .

How can mouse models be used to study IL3RA's role in leukemia?

Mouse models provide valuable insights into IL3RA's role in leukemic development and progression:

• Transgenic approaches:

  • Overexpression of IL3RA in hematopoietic stem cells to mimic high IL3RA expression seen in leukemia stem cells

  • Conditional knockout models to study the requirement of IL3RA in leukemia initiation and maintenance

  • Models with altered IL3RA/βc ratios to investigate the impact of receptor stoichiometry on leukemic transformation

• Xenograft models:

  • Transplantation of human leukemic cells with varying IL3RA/βc ratios into immunodeficient mice (e.g., NSG or NSG-SGM3 mice expressing human IL-3)

  • Limiting dilution assays to assess leukemia stem cell frequency in populations with different IL3RA/βc ratios

• In vitro immortalization models:

  • HoxA9-immortalized IL3RA−/− and βcIL3−/− cells reconstituted with human IL3RA and βc to study receptor assembly and signaling

Research findings show that in acute myeloid leukemia (AML), high IL3RA/βc ratios correlate with stemness and poor survival. In mouse models, fractions with high IL3RA/βc ratios demonstrated 10-240 fold higher leukemia stem cell frequencies compared to fractions with low ratios, depending on the AML sample and mouse model used .

What techniques can be used to study IL3RA receptor assembly and stoichiometry?

Advanced techniques to investigate IL3RA receptor assembly include:

• Fluorescence Resonance Energy Transfer (FRET):

  • Tag IL3RA subunits with donor (e.g., SYFP2) and acceptor (e.g., mScarlet-I) fluorescent proteins

  • Measure changes in FRET efficiency using fluorescence lifetime imaging (FLIM)

  • Detect formation of higher-order receptor complexes based on proximity between fluorescently tagged subunits

  • In published studies, IL-3 stimulation caused a 3.2% ± 0.5% increase in IL3Rα–IL3Rα' FRET efficiency, confirming the formation of higher-order IL3R complexes

• Biochemical approaches:

  • Co-immunoprecipitation to identify receptor subunit interactions

  • Cross-linking followed by SDS-PAGE to capture and analyze receptor complexes

  • Blue native PAGE to preserve native protein complexes and determine their size

• Structural biology:

  • Cryo-electron microscopy to determine 3D structure of receptor complexes

  • X-ray crystallography of receptor components

  • In silico modeling of receptor assemblies based on known structures

• Quantitative flow cytometry:

  • Measure surface expression levels of IL3RA and beta subunits

  • Calculate IL3RA/βc ratios at the protein level

  • Correlate with functional outcomes and receptor assembly

How do I troubleshoot common issues in mouse IL3RA detection by flow cytometry?

When encountering problems with IL3RA detection by flow cytometry:

• High background signal:

  • Issue: Nonspecific binding of antibodies, especially with APC-conjugated antibodies

  • Solution: When using lysing buffers like BD Pharm Lyse™, perform lysis before antibody staining or fix samples with formaldehyde to prevent binding of antibodies to erythroid fragments

  • Alternative: Use different lysing solutions (e.g., BD FACS™ Lysing Solution) after staining

• Weak signal intensity:

  • Issue: Low IL3RA expression or suboptimal antibody concentration

  • Solution: Optimize antibody concentration through titration experiments

  • Alternative: Use signal amplification methods or brighter fluorochromes

• Inconsistent results across experiments:

  • Issue: Variable IL3RA expression or technical variability

  • Solution: Include consistent positive controls (cell lines with known IL3RA expression)

  • Alternative: Normalize to isotype controls and use standardized protocols

• False positive/negative results:

  • Issue: Cross-reactivity or epitope masking

  • Solution: Validate antibody specificity using IL3RA knockout cells or competitive binding assays

  • Alternative: Use multiple antibody clones recognizing different epitopes

What are the key considerations when comparing IL3RA expression data between different mouse strains?

When comparing IL3RA expression across mouse strains:

• Genetic background effects:

  • Different mouse strains can have baseline differences in IL3RA expression and signaling

  • Include strain-matched controls in all experiments

  • Consider backcrossing to a common background when comparing genetically modified models

• Age and sex considerations:

  • IL3RA expression can vary with age and sex in different hematopoietic compartments

  • Match experimental and control groups for age and sex

  • Report age and sex information in all experimental descriptions

• Tissue-specific expression patterns:

  • IL3RA expression varies across hematopoietic compartments

  • Compare the same cell populations when evaluating IL3RA expression

  • Use consistent gating strategies for flow cytometry analysis

• Standardization of measurement techniques:

  • Use the same antibody clones, fluorochromes, and protocols

  • Include calibration beads to normalize fluorescence intensity

  • Calculate relative expression using consistent reference genes for qPCR analysis

• Environmental factors:

  • Housing conditions, microbiome, and pathogen status can affect the immune system

  • Use mice from the same facility when possible

  • Report housing conditions and health status in experimental methods