Recombinant Mouse Late secretory pathway protein AVL9 homolog (Avl9)

What is AVL9 and what are its primary cellular functions?

AVL9 (Late Secretory Pathway Protein AVL9 Homolog) is a protein involved in cell migration and secretory pathway regulation. The protein plays critical roles in post-transcriptional regulation of gene expression and can affect cellular adhesion mechanisms . Recent studies indicate that AVL9 may participate in the negative regulation of vascular endothelial growth factor receptor signaling pathways . In mice, AVL9 shares functional homology with human AVL9, though with some species-specific characteristics in expression patterns and regulatory mechanisms.

Methodologically, researchers investigating AVL9 function should employ both loss-of-function approaches (siRNA knockdown, CRISPR/Cas9 knockout) and gain-of-function strategies (overexpression of recombinant protein) to comprehensively understand its cellular roles.

What experimental systems are optimal for studying recombinant mouse AVL9?

For recombinant expression of mouse AVL9, several experimental systems have proven effective:

When selecting an expression system, researchers should consider that the rBac-SF9 system has demonstrated superior cost performance compared to the HEK293 system for producing recombinant proteins, with potentially 10-fold better efficiency . Expression vectors incorporating the CMV promoter have shown effective expression patterns in multiple systems .

What detection methods are most reliable for mouse AVL9 in tissue samples?

Reliable detection of mouse AVL9 requires appropriate methods based on experimental goals:

For protein detection, western blotting with validated anti-AVL9 antibodies provides quantitative assessment of expression levels, as demonstrated in studies of related proteins . When analyzing tissue distribution, immunohistochemistry with appropriate controls for background autofluorescence is recommended, using techniques such as Sky Blue 6B to eliminate background signals .

For transcript analysis, RT-qPCR has proven effective in detecting AVL9 expression differences between normal and pathological tissue states . Primers targeting conserved regions of the AVL9 gene yield the most consistent results. Additionally, fluorescent reporter systems (such as GFP fusion constructs) can facilitate real-time monitoring of AVL9 expression and localization .

How do expression patterns of mouse AVL9 compare to human AVL9 in normal versus pathological states?

For mouse AVL9, researchers should establish baseline expression profiles across tissue types before examining pathological models. When comparing mouse and human expression patterns, consider:

• Tissue-specific expression profiles in normal states

• Expression changes in disease models (particularly cancer models)

• Correlation between expression levels and disease progression markers

Current data suggests conservation of expression patterns between species, but mouse-specific studies are needed to confirm pathological relevance. Methodologically, researchers should employ matched normal-diseased tissue pairs and standardized quantification protocols for accurate comparisons.

What protein-protein interactions are critical for mouse AVL9 function?

Protein-protein interaction (PPI) analyses of human AVL9 have identified several key interaction partners, including KBTBD2, KIAA1147, EPDR1, and RNF216 . These interactions were positively correlated with AVL9 expression according to Pearson's correlation coefficient analysis .

For mouse AVL9, researchers should:

• Conduct co-immunoprecipitation experiments followed by mass spectrometry to identify interaction partners

• Validate key interactions through techniques such as proximity ligation assays or FRET

• Map interaction domains through truncation or point mutation studies

Understanding the conservation of these interaction networks between mouse and human models is essential for translational research. Current evidence suggests involvement in ubiquitin-mediated proteolysis signaling pathways, indicating potential roles in protein degradation mechanisms .

What regulatory mechanisms control mouse AVL9 expression and activity?

Studies on AVL9 regulatory mechanisms have primarily focused on human models, with limited direct investigation in mouse systems. Current evidence suggests regulation at both transcriptional and post-transcriptional levels.

GO analysis of AVL9 expression-related genes revealed enrichment in post-transcriptional regulation of gene expression . KEGG pathway analysis indicated involvement in several regulatory pathways, including progesterone-mediated signaling, axon guidance, insulin signaling, and ubiquitin-mediated proteolysis .

Researchers investigating mouse AVL9 regulation should:

• Identify transcription factors binding to the AVL9 promoter through ChIP-seq

• Examine potential microRNA regulation through reporter assays

• Investigate post-translational modifications using mass spectrometry

• Assess protein stability and turnover rates under various cellular conditions

Understanding these regulatory mechanisms is crucial for designing experimental interventions that can effectively modulate AVL9 levels or activity in research models.

How can recombinant mouse AVL9 be utilized in therapeutic target validation studies?

Given the emerging role of AVL9 as a potential biomarker and therapeutic target in human colorectal cancer , recombinant mouse AVL9 provides a valuable tool for preliminary target validation in mouse models.

Researchers should consider:

• Developing mouse models with conditional AVL9 expression to evaluate dose-dependent effects

• Using recombinant AVL9 to identify small molecule binders through screening approaches

• Evaluating antibody-based targeting strategies in relevant disease models

• Conducting comparative studies between mouse and human AVL9 to determine conservation of druggable sites

What are the technical challenges in producing functional recombinant mouse AVL9 protein?

Production of functional recombinant mouse AVL9 presents several technical challenges:

Lessons from recombinant protein production systems, such as the rBac system used for viral vectors, indicate that selection of the appropriate production platform significantly impacts yield and functionality . For AVL9, mammalian expression systems may better preserve native conformation and post-translational modifications critical for interaction studies.

How do signaling pathways associated with mouse AVL9 compare across disease models?

Analysis of AVL9-associated signaling pathways revealed connections to several key cellular processes. GO function enrichment analysis showed AVL9 expression-related genes were functionally concentrated in single organismal cell-cell adhesion, post-transcriptional regulation of gene expression, and negative regulation of vascular endothelial growth factor receptor signaling pathway .

KEGG pathway analysis demonstrated involvement in:

• Progesterone-mediated oocyte maturation

• Axon guidance

• Insulin signaling pathway

• Ubiquitin-mediated proteolysis signaling pathways

For mouse models, researchers should employ:

• Phosphoproteomics to map signaling changes following AVL9 modulation

• Pathway-specific reporter assays to quantify pathway activation

• Inhibitor studies to determine pathway dependencies

• Comparative analyses across different disease models (cancer, metabolic disorders, etc.)

Understanding pathway conservation between mouse and human models is essential for translational relevance. Techniques such as RNA-seq and proteomics can provide comprehensive views of pathway alterations in response to AVL9 modulation.

What is the predictive value of mouse AVL9 expression in disease progression models?

For mouse disease models, researchers should:

• Establish baseline expression levels across relevant tissues

• Track expression changes during disease progression

• Correlate expression with established disease markers

• Evaluate interventions targeting AVL9 at different disease stages

Time-course studies are particularly valuable, as demonstrated by GFP reporter studies showing expression patterns over extended periods (14-120 days) . Such longitudinal approaches allow assessment of AVL9's role in disease initiation versus progression.

How can CRISPR/Cas9 technology be optimized for studying mouse AVL9 function?

CRISPR/Cas9 technology offers powerful approaches for investigating AVL9 function through:

• Complete knockout studies to assess developmental and physiological requirements

• Conditional knockout models using Cre-loxP systems for tissue-specific deletion

• Knock-in models for introducing tagged versions or specific mutations

• CRISPRi/CRISPRa systems for reversible modulation of expression levels

When designing CRISPR experiments for mouse AVL9:

Delivery methods such as AAV9-based vectors have shown effective gene transduction in various tissues and could be adapted for delivering CRISPR components . For in vivo applications, careful assessment of delivery efficiency and potential toxicity is essential, similar to evaluations conducted for viral vector systems .