SCGB1A1 Mouse

What is SCGB1A1 and what tissue distribution does it show in mice?

SCGB1A1, also known as Club Cell Secretory Protein (CCSP) or uteroglobin, is primarily expressed in non-ciliated bronchiolar club cells in the lungs. In mice, SCGB1A1 is detected predominantly in bronchial and alveolar cells as demonstrated by lineage tracing studies using Scgb1a1-Cre/Rosa26-tdTomato mice . While the lung shows the strongest expression, some Scgb1a1-driven recombination has been detected in the spleen, suggesting limited expression in extrapulmonary tissues . Notably, SCGB1A1 is a component of pulmonary surfactant and has documented anti-inflammatory properties.

How can researchers confirm SCGB1A1 expression in mouse tissue samples?

Multiple complementary methods can be employed to confirm SCGB1A1 expression:

• Immunohistochemistry/Immunofluorescence: Fixed tissues can be stained using anti-SCGB1A1 antibodies, as demonstrated in studies where paraformaldehyde-fixed lung sections were stained with anti-SCGB1A1 antibodies (R&D Systems) and visualized with fluorophore-conjugated secondary antibodies (Alexa 488 or Alexa 594) .

• RT-PCR/qPCR: RNA isolation followed by reverse transcription and PCR amplification using Scgb1a1-specific primers.

• Reporter mouse models: Utilizing Scgb1a1-IRES-Cre mice crossed with reporter lines like Rosa26-tdTomato allows visualization of cells expressing Scgb1a1 through fluorescent protein expression .

• Western blotting: Protein extraction from lung tissues followed by immunoblotting with anti-SCGB1A1 antibodies.

For accurate interpretation, researchers should include appropriate positive controls (wild-type lung tissue) and negative controls (tissues known not to express SCGB1A1 or samples from Scgb1a1 knockout mice).

What are the primary available SCGB1A1 mouse models for research?

Several mouse models exist for SCGB1A1 research:

• Scgb1a1 knockout (KO) mice: Complete germline deletion of the Scgb1a1 gene (Scgb1a1−/−), useful for studying the physiological role of SCGB1A1 .

• Scgb1a1-IRES-Cre mice: These mice express Cre recombinase from the endogenous Scgb1a1 locus (C57BL/6Smoc-Scgb1a1em1(IRES-Cre)Smoc), enabling conditional gene manipulation in SCGB1A1-expressing cells .

• Inducible Scgb1a1-CreER mice: Allow for temporally controlled recombination specifically in club cells.

• Reporter lines: When crossed with Cre-driver lines, mice such as Rosa26-tdTomato can be used to visualize and track SCGB1A1-expressing cells during development or in disease states .

Each model offers distinct advantages depending on the research question being addressed, from studies of complete SCGB1A1 deficiency to lineage tracing of club cells during development and disease.

How does SCGB1A1 deficiency impact asthma models in mice?

SCGB1A1 deficiency significantly exacerbates asthma phenotypes in mouse models. When challenged with ovalbumin (OVA), Scgb1a1 knockout mice exhibited:

• Increased airway hyperreactivity compared to wild-type mice

• Enhanced inflammatory responses in the lungs

• More severe airway inflammation with increased inflammatory cell infiltration

• Downregulation of FoxA2, a transcriptional regulator also reduced in asthma conditions

These findings confirm the protective, anti-inflammatory role of SCGB1A1 in asthma pathogenesis. The research suggests that diminished SCGB1A1 levels, as observed in human asthma patients, may not merely be a consequence of the disease but actively contribute to worsening respiratory symptoms and inflammation . This provides a mechanistic link between the clinical observation of reduced SCGB1A1 levels in human asthma patients and the functional consequences of this reduction.

What is the relationship between SCGB1A1 and alveolar macrophage function?

SCGB1A1 significantly influences alveolar macrophage (AM) development and inflammatory responses:

• Developmental impact: Transcriptomic analysis revealed that AMs from Scgb1a1-sufficient mice showed upregulation of 37 biological pathways during development from weaning (4 weeks) to early adulthood (12 weeks), with 30 pathways directly involved in antigen presentation, anti-viral immunity, and inflammation .

• Inflammatory regulation: Under Scgb1a1 deficiency, these immune-related pathways were significantly downregulated compared to age-matched Scgb1a1-sufficient mice .

• Early inflammation: AMs from Scgb1a1-deficient mice demonstrated premature activation of inflammatory pathways compared to wild-type counterparts .

• Cytokine modulation: In vitro experiments demonstrated that SCGB1A1 protein supplementation significantly reduced AM responses to microbial stimuli, blunting the release of pro-inflammatory cytokines and chemokines including IL-1β, IL-6, IL-8, MIP-1α, TNF-α, and MCP-1 after TLR stimulation .

These findings suggest SCGB1A1 plays a crucial role in shaping AM-mediated inflammation and immune responses, particularly in preventing excessive inflammatory responses to stimuli. This represents a novel mechanism by which club cells may influence lung immunity through secreted SCGB1A1.

How does SCGB1A1 expression change during respiratory infections or inflammatory conditions?

During respiratory infections and inflammatory conditions, SCGB1A1 expression is significantly altered:

• Viral infections: Human rhinovirus infection reduces expression of both SCGB1A1 and its transcriptional regulator FOXA2 in epithelial cells .

• Th2 inflammation: Pro-allergic Th2 cytokines (IL-4 and IL-13) repress epithelial expression of SCGB1A1 and FOXA2, linking allergic inflammation to reduced SCGB1A1 levels .

• Asthma: Analysis of multiple gene expression datasets revealed that SCGB1A1 mRNA is significantly reduced (by approximately 53%) in epithelial cells from asthma patients compared to healthy controls, while the mucin gene MUC5AC is elevated approximately twofold .

• Age-related changes: In Scgb1a1-sufficient mice, AM activation of immune pathways increases from weaning to early adulthood, but this development is impaired in Scgb1a1-deficient mice .

These expression changes suggest SCGB1A1 functions in a complex regulatory network that responds to environmental challenges. The reduction in SCGB1A1 during inflammatory conditions may contribute to disease exacerbation by removing a key anti-inflammatory mediator.

What are the optimal strategies for inducing experimental asthma in SCGB1A1 mouse models?

For robust experimental asthma induction in SCGB1A1 mouse models, researchers should consider the following protocol elements:

• Ovalbumin (OVA) sensitization and challenge:

  • Sensitization: Intraperitoneal injection of OVA with aluminum hydroxide adjuvant on days 0 and 14

  • Challenge: Aerosolized OVA exposure (1% in PBS) for 30 minutes on days 28, 29, and 30

• Alternative models:

  • House dust mite (HDM) extract: Intranasal administration of HDM (25 μg) on days 0, 7, and 14-18

  • IL-13 instillation: Direct administration of recombinant IL-13 to model Th2-high asthma phenotypes

• Readout parameters:

  • Airway hyperreactivity using whole-body plethysmography or forced oscillation techniques

  • Bronchoalveolar lavage (BAL) cell differential counts

  • Histopathological analysis of lung sections with H&E, PAS, and Masson's trichrome staining

  • Immunostaining for SCGB1A1 and MUC5AC to assess club cell integrity and mucus production

• Controls:

  • Age and sex-matched wild-type littermates

  • Vehicle-only challenges

  • Both Scgb1a1-sufficient and Scgb1a1-deficient mice should be included to determine the role of SCGB1A1

This comprehensive approach allows for detailed characterization of how SCGB1A1 deficiency influences asthma pathogenesis and provides a platform for testing potential therapeutic interventions.

How can researchers effectively use Scgb1a1-IRES-Cre mice for lineage tracing studies?

Effective lineage tracing with Scgb1a1-IRES-Cre mice requires careful experimental design:

• Cross with appropriate reporter lines:

  • Rosa26-tdTomato reporter mice provide strong red fluorescence in cells expressing Cre

  • Rosa26-mTmG reporters allow visualization of membrane-targeted GFP in recombined cells

  • Rosa26-Confetti enables multicolor lineage tracing for clonal analysis

• Validation of Cre activity:

  • Confirm recombination efficiency through direct fluorescence microscopy

  • Analyze tissue-specific recombination patterns (in lung, Scgb1a1-Cre drives recombination in bronchial and alveolar cells)

  • Test for off-target recombination in other tissues (some recombination has been detected in the spleen with Scgb1a1-Cre)

• Temporal studies:

  • Perform analyses at multiple timepoints to track cell fate

  • For developmental studies, examine embryonic, neonatal, and adult stages

  • For injury models, establish baseline recombination before injury and follow recovery

• Technical considerations:

  • Use co-staining with cell-specific markers to confirm identity of lineage-traced cells

  • Employ confocal microscopy with Z-stack imaging for three-dimensional analysis

  • Consider flow cytometry for quantitative assessment of lineage-traced populations

• Controls:

  • Include Cre-negative littermates

  • Use alternative Cre driver lines to validate findings (e.g., other club cell-targeting Cre lines)

This approach allows researchers to trace the fate of Scgb1a1-expressing cells during development, homeostasis, and disease conditions, providing insights into club cell dynamics and potential contribution to epithelial regeneration.

What methods are recommended for isolating and culturing primary SCGB1A1-expressing club cells?

Isolating and maintaining primary club cells requires specialized techniques:

• Isolation procedures:

  • Bronchioalveolar lavage: For collecting superficial club cells (limited yield)

  • Enzymatic digestion: Using dispase/collagenase treatment of lung tissue

  • Flow cytometry: Sorting based on Scgb1a1-driven fluorescent reporter expression

  • Magnetic bead separation: Using antibodies against club cell surface markers

• Culture conditions:

  • Media composition: DMEM/F12 supplemented with insulin, transferrin, selenium, hydrocortisone, EGF, bovine pituitary extract, and retinoic acid

  • Substratum: Growth on collagen-coated surfaces or air-liquid interface cultures

  • 3D organoid culture: Mixed with Matrigel to form bronchospheres

  • Co-culture systems: With fibroblasts to better mimic the in vivo niche

• Verification of cell identity:

  • Immunostaining for SCGB1A1 protein

  • qPCR for Scgb1a1 and other club cell markers

  • Absence of markers for ciliated cells, basal cells, and neuroendocrine cells

  • Functional secretory capacity assessment

• Experimental applications:

  • Cytokine stimulation: IL-4/IL-13 treatment to model Th2 inflammation

  • Viral infection: Human rhinovirus to study infection responses

  • Gene manipulation: siRNA or CRISPR-Cas9 for targeted gene knockdown/knockout

  • Drug testing: Evaluating responses to therapeutics targeting inflammatory pathways

These methods enable the study of primary SCGB1A1-expressing cells in controlled environments, allowing detailed investigation of their responses to various stimuli and potential therapeutic interventions.

How can SCGB1A1 mouse models be utilized to study airway regeneration after injury?

SCGB1A1 mouse models offer valuable tools for studying airway regeneration:

• Lineage tracing approaches:

  • Scgb1a1-IRES-Cre or Scgb1a1-CreER mice crossed with reporter lines (Rosa26-tdTomato or Rosa26-Confetti) allow tracking of club cell fate during regeneration

  • Pulse-chase experiments with inducible Scgb1a1-CreER systems can distinguish pre-existing from newly differentiated club cells

• Injury models compatible with SCGB1A1 studies:

  • Naphthalene injury: Targets club cells through cytochrome P450-2F2 metabolism

  • Influenza infection: Causes widespread epithelial damage

  • Bleomycin: Induces fibrotic responses following epithelial injury

  • Ozone exposure: Models oxidative damage to the airway epithelium

• Analytical approaches:

  • Single-cell RNA sequencing to characterize regenerative cell populations

  • Spatial transcriptomics to map regeneration zones

  • Time-course analysis of SCGB1A1 re-expression during repair

  • Co-localization studies of SCGB1A1 with proliferation markers (Ki67, BrdU)

• Mechanistic investigations:

  • Conditional knockout of regeneration-associated genes in SCGB1A1+ cells

  • Selective ablation of SCGB1A1+ cells to assess their contribution to repair

  • Exogenous SCGB1A1 administration to determine if it accelerates regeneration

These approaches can reveal the contribution of club cells to epithelial maintenance and regeneration, which has implications for understanding chronic lung diseases characterized by impaired repair, such as COPD and pulmonary fibrosis.

What insights have SCGB1A1 mouse models provided about the club cell's role in immune modulation?

SCGB1A1 mouse models have revealed critical immunomodulatory functions of club cells:

• Alveolar macrophage programming:

  • SCGB1A1 influences the developmental trajectory of alveolar macrophages (AMs)

  • Transcriptomic analysis showed that Scgb1a1 sufficiency is associated with upregulation of 37 biological pathways in AMs during development from weaning to adulthood, with 30 pathways directly involved in antigen presentation, anti-viral immunity, and inflammation

  • Under Scgb1a1 deficiency, these immune-related pathways were significantly downregulated compared to wild-type mice

• Cytokine regulation:

  • SCGB1A1 protein supplementation significantly reduced AM responses to microbial stimuli in vitro

  • SCGB1A1 blunted the release of multiple pro-inflammatory cytokines and chemokines including IL-1β, IL-6, IL-8, MIP-1α, TNF-α, and MCP-1 after TLR stimulation

  • This suggests SCGB1A1 acts as a natural brake on inflammatory responses in the lung

• Response to pathogen-associated molecular patterns:

  • SCGB1A1 modulates AM responses to various TLR agonists including:

    • TLR2 ligands (heat-killed Listeria monocytogenes)

    • TLR4 ligands (LPS from Escherichia coli K12)

    • TLR5 ligands (Salmonella typhimurium Flagellin)

• Age-dependent immune regulation:

  • AMs from Scgb1a1-deficient mice showed premature activation of inflammatory pathways compared to wild-type counterparts

  • This suggests SCGB1A1 helps maintain appropriate timing of immune maturation in the lung

These findings collectively establish SCGB1A1 as a critical mediator in club cell-macrophage crosstalk and highlight the importance of epithelial-derived factors in shaping lung immune responses.

What methodological approaches are recommended when investigating SCGB1A1 protein function in vitro?

Effective investigation of SCGB1A1 protein function requires specialized methodological approaches:

• Protein sources and preparation:

  • Recombinant SCGB1A1 protein (commercially available or lab-produced)

  • Recommended working concentration: 5 μg/mL for in vitro studies

  • Purification from BAL fluid of wild-type mice

  • Expression in mammalian cell systems for proper post-translational modifications

• Cellular models:

  • Primary alveolar macrophages isolated by BAL and flow sorting (CD11c^hi Siglec-F^hi)

  • Human bronchial epithelial cells (HBECs) for studies on SCGB1A1 expression and regulation

  • Air-liquid interface cultures to mimic physiological conditions

  • Precision-cut lung slices to maintain cell-cell interactions

• Functional assays:

  • Inflammation studies:

    • Treatment of cells with TLR agonists (LPS, HKLM, Flagellin) with/without SCGB1A1

    • Measure cytokine/chemokine release via multiplex immunoassays

    • Assess inflammatory pathway activation through phospho-protein analysis

  • Gene regulation studies:

    • Analysis of FOXA2-dependent SCGB1A1 expression

    • Promoter-reporter assays to study transcriptional regulation

    • ChIP assays to identify transcription factor binding sites

• Molecular interaction studies:

  • Co-immunoprecipitation to identify SCGB1A1 binding partners

  • Surface plasmon resonance to measure binding kinetics

  • Confocal microscopy to track cellular uptake and localization

• Controls and validation:

  • Heat-inactivated SCGB1A1 protein as negative control

  • Dose-response curves to establish optimal concentrations

  • Comparison with other secretoglobin family members

  • Knockdown/knockout validation in relevant cell types

These methodological approaches provide a comprehensive framework for investigating SCGB1A1's functions in inflammation regulation, cellular protection, and immune modulation in controlled experimental systems.

What evidence supports SCGB1A1 as a potential therapeutic agent for inflammatory lung diseases?

Multiple lines of evidence support SCGB1A1's therapeutic potential:

• Anti-inflammatory effects:

  • Exogenous SCGB1A1 protein supplementation significantly reduced cytokine and chemokine production by alveolar macrophages in response to TLR agonists

  • SCGB1A1 blunted the release of IL-1β, IL-6, IL-8, MIP-1α, TNF-α, and MCP-1, all key mediators in lung inflammation

• Protection in asthma models:

  • Scgb1a1 knockout mice showed exacerbated airway hyperreactivity and inflammation when exposed to ovalbumin, confirming SCGB1A1's protective role

  • FOXA2 overexpression restored repressed SCGB1A1 expression in IL-13-treated or rhinovirus-infected cells, suggesting potential therapeutic pathways

• Mitigation of cytokine responses:

  • SCGB1A1 effectively mitigated cytokine surges in the lungs, which could be beneficial in conditions characterized by excessive inflammation

  • This property is particularly relevant for diseases like acute respiratory distress syndrome or severe COVID-19

• Clinical correlations:

  • Lower levels of SCGB1A1 are consistently found in BAL fluid, serum, and sputum from asthma patients

  • SCGB1A1 levels are also reduced in COPD patients and correlate with lung function decline

  • These clinical associations support the concept that restoring SCGB1A1 levels might ameliorate disease

• Emerging applications:

  • SCGB1A1 has been identified as a potential biomarker and therapeutic target for head and neck squamous cell carcinoma (HNSCC)

These findings collectively suggest that recombinant SCGB1A1 administration or strategies to enhance endogenous SCGB1A1 expression could represent novel therapeutic approaches for inflammatory lung diseases and potentially other conditions.

How do findings from SCGB1A1 mouse models translate to human respiratory diseases?

Translating findings from SCGB1A1 mouse models to human disease reveals important parallels and considerations:

• Expression patterns and reductions in disease:

  • Both humans and mice show reduced SCGB1A1 levels in asthma

  • Human SCGB1A1 protein is significantly reduced in BAL, sputum, and serum from asthma patients compared to healthy individuals

  • Similarly, mouse models show Scgb1a1 downregulation in ovalbumin-induced asthma

• Genetic aspects:

  • The human SCGB1A1 gene has a polymorphism (A38G, rs3741240) associated with asthma development and severity

  • The A allele correlates with reduced SCGB1A1 levels in BAL and circulation, mirroring the mouse phenotype

• Regulatory mechanisms:

  • FOXA2 regulation of SCGB1A1 appears conserved between species

  • Both human and mouse show IL-13 and rhinovirus-induced repression of SCGB1A1 and FOXA2

• Functional outcomes:

  • The anti-inflammatory properties of SCGB1A1 observed in mouse models likely apply to humans

  • SCGB1A1's ability to attenuate airway inflammation and delay lung function decline has been demonstrated in smoking-induced models relevant to COPD

• Translational challenges:

  • Species differences in club cell distribution and abundance must be considered

  • Mouse models may not fully recapitulate the chronic nature of human respiratory diseases

  • Human genetic diversity creates variability not captured in inbred mouse strains

These translational insights provide a framework for developing SCGB1A1-based therapeutic strategies that could be effective in human disease, while highlighting important considerations for proper interpretation of mouse model findings.

What approaches can be used to target or enhance SCGB1A1 expression for therapeutic purposes?

Multiple strategic approaches can target or enhance SCGB1A1 expression:

• Direct protein delivery:

  • Recombinant SCGB1A1 protein administration via aerosolization

  • Lipid nanoparticle encapsulation for improved stability and delivery

  • PEGylation to increase half-life in the respiratory tract

  • Development of SCGB1A1 analogues with enhanced stability or function

• Transcriptional enhancement:

  • FOXA2 augmentation strategies, as FOXA2 has been shown to drive promoter activity and expression of SCGB1A1

  • FOXA2 overexpression restored repressed SCGB1A1 expression in IL-13–treated or rhinovirus-infected cells

  • Small molecule screening to identify compounds that enhance SCGB1A1 promoter activity

• Gene therapy approaches:

  • AAV-mediated delivery of SCGB1A1 expression cassettes

  • CRISPR activation systems targeting the endogenous SCGB1A1 promoter

  • Club cell-targeted delivery using Scgb1a1 promoter-driven constructs

• Epigenetic modulation:

  • Histone deacetylase inhibitors to promote chromatin accessibility at the SCGB1A1 locus

  • DNA methyltransferase inhibitors if SCGB1A1 is silenced by methylation in disease states

• Pathway-based approaches:

  • Antagonism of IL-4/IL-13 signaling to prevent Th2-mediated SCGB1A1 repression

  • Anti-viral strategies to prevent rhinovirus-induced SCGB1A1 downregulation

  • TLR pathway modulators to leverage SCGB1A1's role in regulating macrophage responses

• Combination therapies:

  • SCGB1A1 enhancement combined with anti-inflammatory corticosteroids

  • SCGB1A1 with bronchodilators for asthma and COPD applications

  • Sequential therapy targeting acute inflammation followed by SCGB1A1-based tissue repair

These approaches offer multiple avenues for therapeutic development, with selection depending on the specific disease context, delivery challenges, and desired functional outcomes.

What are common challenges when working with SCGB1A1 mouse models and how can they be addressed?

Researchers commonly encounter several challenges when working with SCGB1A1 mouse models:

• Phenotypic variability:

  • Challenge: Inconsistent phenotypes between studies or mouse colonies

  • Solution: Maintain consistent genetic background through backcrossing; report complete strain information; use littermate controls; standardize housing conditions and microbiome status

• Off-target effects in Cre models:

  • Challenge: Scgb1a1-Cre expression in non-targeted tissues (e.g., detected in spleen)

  • Solution: Thoroughly characterize Cre expression patterns with reporter lines before experimental interpretation; include Cre-only controls; validate findings with alternative club cell targeting approaches

• Developmental compensation:

  • Challenge: Germline Scgb1a1 knockout mice may develop compensatory mechanisms

  • Solution: Use inducible knockout systems; compare acute vs. chronic deletion effects; examine expression of other secretoglobin family members

• Technical detection issues:

  • Challenge: Difficulty in SCGB1A1 protein detection or quantification

  • Solution: Optimize fixation protocols for immunohistochemistry (4% paraformaldehyde recommended) ; use validated antibodies (R&D Systems anti-SCGB1A1) ; consider multiple detection methods (ELISA, Western blot, immunofluorescence)

• Disease model variability:

  • Challenge: Inconsistent responses to asthma induction or other challenges

  • Solution: Standardize protocols for allergen sensitization and challenge; consider sex as a biological variable; control for age effects (demonstrated importance of age in SCGB1A1 studies)

• Translational limitations:

  • Challenge: Differences between mouse and human club cell biology

  • Solution: Validate key findings in human samples or cell systems; acknowledge species differences in result interpretation; consider using humanized mouse models when appropriate

Addressing these challenges requires careful experimental design, proper controls, and transparent reporting of methodological details to ensure reproducibility and translatability of findings.

How should researchers interpret contradictory findings in SCGB1A1 mouse studies?

When faced with contradictory findings in SCGB1A1 mouse studies, researchers should employ a systematic approach to reconciliation:

• Methodological differences assessment:

  • Mouse strain variations: Different genetic backgrounds can significantly influence phenotypes

  • Model specifics: Compare knockout strategies (germline vs. conditional), Cre driver lines, and reporter systems

  • Age considerations: SCGB1A1 functions change with development—compare if studies used mice of different ages

  • Sex differences: Determine if contradictions could be explained by male vs. female differences

• Experimental context evaluation:

  • Disease induction protocols: Variations in allergen dose, route, adjuvant, and challenge schedule

  • Environmental factors: Housing conditions, microbiome composition, and pathogen status

  • Timing of analysis: Acute vs. chronic responses may differ substantially

• Molecular mechanism investigation:

  • Pathway redundancy: Consider if alternative pathways compensate differently in various models

  • FOXA2 relationship: Examine if discrepancies relate to FOXA2 levels, as FOXA2 regulates SCGB1A1

  • Inflammation context: The specific inflammatory milieu (Th1 vs. Th2) may alter SCGB1A1 effects

• Reconciliation strategies:

  • Direct replication studies: Using standardized protocols across laboratories

  • Meta-analysis approaches: Systematically analyzing multiple datasets, as done for human SCGB1A1 expression in asthma

  • Combinatorial experiments: Simultaneously testing multiple variables to identify interaction effects

  • Single-cell approaches: Determining if contradictions reflect differences in specific cell populations

• Translational relevance assessment:

  • Compare mouse findings with human data when available

  • Consider if contradictions reflect the heterogeneity also seen in human disease

This structured approach can help researchers navigate apparent contradictions and extract meaningful biological insights from seemingly discrepant results in the SCGB1A1 literature.

What quality control measures are essential when breeding and maintaining SCGB1A1 mouse lines?

Maintaining high-quality SCGB1A1 mouse lines requires rigorous quality control measures:

• Genotyping protocols:

  • Regular PCR-based genotyping using primers specific to wild-type and modified Scgb1a1 alleles

  • For Scgb1a1-Cre lines, additional primers to detect Cre recombinase

  • Periodic verification of genotyping accuracy through alternative methods

  • Include positive and negative controls with every genotyping batch

• Expression verification:

  • Periodic confirmation of SCGB1A1 protein expression (or absence) through immunohistochemistry of lung sections

  • For Cre driver lines, validation of recombination patterns using reporter crosses

  • qPCR assessment of Scgb1a1 transcript levels in lung tissue

  • Western blot analysis of SCGB1A1 protein in BAL fluid

• Colony management:

  • Maintain detailed pedigree records to monitor inbreeding

  • Regular backcrossing to foundational strains (typically every 10 generations)

  • Cryopreservation of embryos from early generations as backup

  • Periodic health monitoring for pathogens that could affect lung phenotypes

• Experimental consistency:

  • Standardize age for experiments based on developmental SCGB1A1 expression patterns

  • Consider sex as a biological variable in all experiments

  • Maintain consistent environmental conditions (temperature, humidity, light cycles)

  • Control for housing density and cage position effects

• Phenotypic drift monitoring:

  • Regular benchmark testing of key phenotypes (e.g., baseline lung function, club cell numbers)

  • Periodic comparison to historical data to detect subtle phenotypic drift

  • Sentinel monitoring for environmental changes that could affect phenotype

• Documentation standards:

  • Detailed record-keeping of breeding performance and health status

  • Documentation of any unexpected phenotypes or breeding challenges

  • Transparency in reporting colony management practices in publications