LGALS7 Mouse

What is the genomic location and basic structure of the LGALS7 gene in mice?

The mouse LGALS7 gene is located on chromosome 19q13.2, analogous to its human counterpart. The full-length cDNA encodes a galectin-7 protein comprised of 136 amino acids . The protein structure contains a characteristic carbohydrate recognition domain (CRD) that is essential for its functionality in binding to glycosylated substrates. When investigating LGALS7 in mice, researchers should note that the protein sequence spans from Ser2 to Phe136 according to standard reference databases (Accession # AAK29385) . The promoter region, which is critical for expression regulation, contains several polymorphic sites that have been associated with pathological conditions in translational studies.

How can LGALS7 expression be manipulated in mouse models for research purposes?

Multiple approaches exist for modulating LGALS7 expression in mice. Overexpression models can be generated through microinjection of pcDNA3.1-LGALS7 plasmid vectors into fertilized oocytes (one-cell-stage fertilized eggs) . For knockdown studies, researchers have successfully used pSilencer-LGALS7 plasmid vectors containing shRNA constructs targeting LGALS7 . Both transgenic lines can be maintained by backcrossing with BALB/c mice.

Genotyping of transgenic founders and offspring should be performed using PCR amplification of tail snip DNA with transgene-specific primers. Validation of successful genetic manipulation can be confirmed at both the transcript level (via PCR) and protein level (via Western blotting) . When designing these experiments, researchers should consider:

For phenotypic validation, expression analysis in target tissues is essential, as shown in transgenic models where differential expression patterns were confirmed through PCR and protein detection methods .

How should experiments be designed to investigate LGALS7's impact on T cell populations in tumor microenvironments?

When investigating LGALS7's effect on T cell populations, a multi-tiered experimental approach is recommended. Based on recent findings, galectin-7 reduces CD4+ T cell percentages while potentially increasing CD8+ T cells in both in vitro and in vivo systems .

For in vitro assessment, human PBMCs should be cultured with purified recombinant galectin-7 (recommended concentration range: 1-10 μg/ml) or BSA control for 7 days. Flow cytometry analysis should be performed to determine the relative percentages of CD4+ and CD8+ T cells among total lymphocytes, using appropriate fluorochrome-conjugated antibodies against CD3, CD4, and CD8 .

For in vivo validation, two complementary approaches can be implemented:

• Humanized mouse models: Inject human PBMCs into NSG mice and treat with galectin-7 (e.g., every 4 days starting from day 14 after PBMC injection). Compare with PBS-treated controls.

• Syngeneic tumor models: Use mouse cancer cell lines (e.g., MC38 colon cancer or LL/2 lung cancer) with either:

  • Exogenous galectin-7 administration (1.5 mg/kg every 4 days)

  • Engineered overexpression of galectin-7 in the cancer cells

Critically, researchers should include both wild-type and specialized knockout models (e.g., PD-1 KO mice) to address mechanistic questions. Endpoints should include analysis of T cell subpopulations in peripheral blood, spleen, and tumor tissues by flow cytometry, with particular attention to CD4+ T cells, CD4+CD25+ regulatory T cells, and CD8+ T cells .

What are the optimal methods for investigating LGALS7 promoter polymorphisms in relation to disease susceptibility?

Investigation of LGALS7 promoter polymorphisms requires careful genetic analysis methodology. Based on recent studies exploring ICH susceptibility, researchers should:

• Define clear case and control populations with detailed inclusion/exclusion criteria

• Extract genomic DNA using standardized protocols

• Sequence the entire LGALS7 promoter region, particularly focusing on known SNP locations (e.g., rs567785577 and rs138945880 on 19q13.2)

• Employ multiple genetic models for analysis:

  • Dominant model (MM vs. Mm + mm)

  • Recessive model (mm vs. Mm + MM)

  • Overdominant model (MM + mm vs. Mm)

  • Codominant model (MM vs. Mm vs. mm)

  • Additive and multiplicative models

Statistical analysis should include calculation of odds ratios with 95% confidence intervals and appropriate correction for multiple testing. Continuous variables should be presented as mean ± SE, with p-values < 0.05 considered statistically significant . For comprehensive pathway analysis, researchers should consider conducting differential protein expression studies, as demonstrated in the transgenic mouse models where iTRAQ peptide analysis identified 1,009 differentially expressed proteins with 28 known proteins showing significant correlation with LGALS7 expression levels .

How does galectin-7 influence tumor growth in different experimental contexts and what are the underlying mechanisms?

The relationship between galectin-7 and tumor growth is context-dependent and requires careful experimental design to elucidate. Based on syngeneic mouse models, galectin-7 demonstrates distinct effects depending on the immune competence of the host and molecular context.

In wild-type immunocompetent mice, galectin-7 administration (1.5 mg/kg every 4 days) suppressed MC38 tumor growth, correlating with a reduction in CD4+ T cells and CD4+CD25+ regulatory T cells . This effect appears to be dependent on PD-1 signaling, as galectin-7 treatment did not reduce CD4+ T cell percentages or suppress tumor growth in PD-1 knockout mice .

The proposed mechanism involves:

• Selective reduction of CD4+ T cells (including regulatory T cells)

• Relative sparing of CD8+ T cells

• De-suppression of anti-tumor immune responses

• Dependence on intact PD-1 signaling pathways

These differential effects highlight the importance of considering both the tumor microenvironment and systemic immune context when investigating galectin-7's role in cancer progression .

What protein interactions and molecular pathways are modulated by LGALS7 expression in mice?

Proteomic analysis of LGALS7 transgenic and knockdown mice has revealed complex molecular networks influenced by this protein. Using iTRAQ peptide identification and mass spectrometry, researchers identified 1,009 differentially expressed proteins associated with galectin-7 expression levels .

Key protein interaction changes observed in LGALS7 knockdown mice include:

• Relatively increased expression:

  • Serum amyloid A protein

  • Actin 1

• Relatively decreased expression:

  • Myosin regulatory light chain 2

  • Keratin (type I cytoskeletal 13)

  • Troponin C

  • Creatine kinase M-type

  • Myosin light chain 2

  • Myosin, heavy polypeptide 8

Conversely, LGALS7 overexpression progressively increased:

• Calsequestrin

• Myosin light chain 1/3

• Myoglobin

• Myosin regulatory light chain 2

• Myosin-1

• Keratin (type II cytoskeletal 4)

Pathway analyses revealed that these protein changes affect:

• Signal transduction cascades

• Molecular metabolic processes

• Cerebrovascular functions

• Blood-brain barrier permeability

• Inflammatory responses, particularly through SAA-mediated chemotactic recruitment of inflammatory cells

These interactions suggest that LGALS7 influences multiple pathways including actin-driven angiogenesis, endothelial cell activities, and vascular basement membrane integrity. The downstream effects include potential impacts on cerebral amyloid angiopathy (CAA) and inflammatory processes around blood vessels .

What are the optimal methods for detecting galectin-7 protein in mouse tissues?

Detection of galectin-7 in mouse tissues requires appropriate methodological approaches depending on the research question. Immunohistochemistry (IHC) has been successfully employed for tissue localization studies, particularly in skin sections where galectin-7 expression is prominent .

Recommended IHC Protocol for Galectin-7 Detection:

• Fix tissue samples (e.g., mouse skin) using immersion fixation in formaldehyde

• Process and embed in paraffin following standard protocols

• Prepare sections (typically 4-6 μm thickness)

• Perform heat-induced epitope retrieval using basic retrieval reagents (pH 9.0)

• Block endogenous peroxidase activity

• Incubate with primary antibody against mouse galectin-7 (optimal concentration: 1 μg/ml) for 1 hour at room temperature

• Use HRP-polymer antibody detection systems for visualization

• Develop with DAB (brown) and counterstain with hematoxylin (blue)

• Evaluate staining patterns, noting that galectin-7 localizes to both cytoplasm and nucleus

For quantitative protein analysis, Western blotting provides reliable results using antibodies with confirmed specificity against mouse galectin-7 . Flow cytometry can also be employed for detection of galectin-7 in cell suspensions, particularly when analyzing interactions with immune cell populations .

Importantly, researchers should consider that galectin-7 expression is tissue-specific, with high expression in epithelial tissues, particularly the skin. When analyzing other tissues, appropriate positive and negative controls should be included to validate detection methods .

How can researchers effectively analyze the functional relationship between LGALS7 and immune cell populations?

To analyze the functional relationship between LGALS7 and immune cell populations, researchers should implement a multi-faceted approach:

• In vitro T cell assays:

  • Culture purified CD4+ and CD8+ T cells with recombinant galectin-7

  • Perform apoptosis assays using activated T cells isolated from human PBMCs via magnetic bead-based sorting

  • Analyze changes in apoptosis markers (e.g., Annexin V/PI staining) by flow cytometry

• In vivo immune composition analysis:

  • Use syngeneic mouse tumor models with galectin-7 treatment (1.5 mg/kg every 4 days)

  • At defined endpoints, harvest peripheral blood, spleen, and tumor tissues

  • Perform comprehensive flow cytometry analysis of immune cell populations

  • Focus on CD4+ T cells, CD8+ T cells, and CD4+CD25+ regulatory T cells

• Genetic manipulation approaches:

  • Compare wild-type mice with PD-1 knockout mice to assess signaling dependencies

  • Consider using cell lines engineered to overexpress galectin-7 for mechanistic studies

  • Test effects in both immunocompetent and immunodeficient backgrounds

• Immune modulation experiments:

  • Combine galectin-7 treatment with immune checkpoint inhibitors (e.g., anti-PD-1 antibodies)

  • Assess cooperative or antagonistic effects on tumor growth and immune populations

  • Dosing regimen: anti-mouse PD-1 at 5 mg/kg by retro-orbital injection, with galectin-7 at 1.5 mg/kg

These approaches should be complemented with appropriate controls, including PBS vehicle controls and isotype control antibodies. Statistical analysis should account for biological variability, with experiments performed in triplicate at minimum.

How can researchers reconcile contradictory findings regarding LGALS7's role in cancer progression?

The contradictory findings regarding LGALS7's role in cancer progression reflect its context-dependent functions and require careful interpretation. Researchers should consider several key factors when reconciling seemingly contradictory data:

• Immune context dependency:

  • In immunocompetent hosts, galectin-7 suppresses tumor growth by modulating CD4+ T cell populations, particularly regulatory T cells

  • In immunodeficient hosts (e.g., NSG mice), galectin-7 overexpression enhances tumor growth, suggesting direct pro-tumorigenic effects on cancer cells

• Signaling pathway dependencies:

  • Galectin-7's tumor-suppressive effects are abolished in PD-1 knockout mice

  • This suggests that intact PD-1 signaling is required for galectin-7's immunomodulatory effects

• Cancer type specificity:

  • Different cancers may exhibit variable responses to galectin-7 based on their intrinsic immune infiltration patterns

  • In tumors with reduced regulatory T cell infiltration, galectin-7 may be less effective at restoring anti-tumor immune responses

• Local glycome composition:

  • Variations in the glycosylation patterns within different tumor microenvironments may affect galectin-7 binding capabilities

  • The presence of competing galectin-7 binding proteins can influence its functional outcomes

To address these contradictions methodologically, researchers should:

• Design experiments with multiple cancer models

• Include both immunocompetent and immunodeficient backgrounds

• Analyze detailed immune cell profiles in tumor microenvironments

• Consider glycomic analyses alongside proteomic approaches

• Implement genetic manipulation studies (knockdown, overexpression, and domain mutants) to dissect mechanism

What are the methodological considerations for interpreting differential protein expression data in LGALS7 transgenic mouse models?

• Sample preparation standardization:

  • Process samples at minimum in triplicate

  • Implement a sandwich-based method with gradient elution for HPLC separation

  • Require peptides to be identified at least twice before inclusion in analysis

• Data filtering and validation:

  • From the initial identification of differentially expressed proteins (e.g., 1,009 proteins in referenced studies), apply stringent filtering

  • Verify key findings with orthogonal techniques (Western blotting, immunohistochemistry)

  • Consider both direct binding partners and downstream effectors

• Biological pathway analysis:

  • Perform enrichment analysis using Gene Ontology (GO) terms

  • Identify enriched functional categories (e.g., signal transduction, metabolic processes)

  • Conduct KEGG pathway mapping to place findings in biological context

• Result interpretation framework:

Researchers should be cautious about several factors that could influence interpretation, including potential off-target effects of genetic manipulations, compensatory mechanisms in transgenic models, and the influence of developmental timing on observed phenotypes .