Recombinant Chicken Multivesicular body subunit 12A (FAM125A)

What is Chicken MVB12A and what cellular functions does it perform?

MVB12A (Multivesicular Body Subunit 12A) is a protein-coding gene that functions as a component of the ESCRT-I complex, which regulates vesicular trafficking processes. In chickens, as in other vertebrates, MVB12A enables lipid binding and ubiquitin binding activities, playing critical roles in:

• Regulation of epidermal growth factor receptor signaling pathways

• Viral budding and virus maturation

• Membrane trafficking and protein sorting

MVB12A protein is located in several cellular components, including the Golgi apparatus, centrosome, and nucleoplasm . The protein functions within the ESCRT (Endosomal Sorting Complex Required for Transport) machinery, which is critical for multivesicular body biogenesis and receptor downregulation, with the complex being highly conserved from yeast to humans .

How do I design primers for chicken MVB12A gene amplification in qRT-PCR experiments?

When designing primers for chicken MVB12A, follow these methodological steps:

• Obtain the chicken MVB12A sequence from genomic databases such as Ensembl (Gallus gallus 5.0, http://asia.ensembl.org/Gallus_gallus/Info/Index)

• Design primers with the following specifications:

  • 18-22 nucleotides in length

  • GC content between 40-60%

  • Melting temperature (Tm) of 58-62°C

  • Amplicon size of 80-150 bp for optimal qRT-PCR efficiency

  • Span exon-exon junctions to avoid genomic DNA amplification

• Validate primer specificity using tools like BLAST against the chicken genome

• Test primers experimentally with a standard curve to confirm amplification efficiency (should be 90-110%)

For RNA extraction and cDNA synthesis, follow protocols similar to those used in gene expression studies of chicken muscle tissue, using 1 μg of total RNA for cDNA synthesis with commercial kits (e.g., Vazyme, Nanjing, China) .

What expression patterns of MVB12A are observed in different chicken tissues?

MVB12A expression in chickens varies across tissue types and developmental stages. Based on current research methodologies:

• Use RNA-seq analysis on multiple tissue types (breast muscle, liver, adipose, etc.)

• Normalize expression values using established housekeeping genes (GAPDH, β-actin)

• Validate with qRT-PCR across different developmental timepoints

In studies of chicken gene expression profiling, breast muscle tissue has been extensively analyzed, particularly during early embryonic development. The methodological approach includes:

• Sampling tissues at specific developmental timepoints

• RNA isolation using TRIzol reagent

• Library construction with paired-end sequencing

• Genome alignment using tools like HISAT2-build (v2.0.4)

• Assembly of mapped reads using StringTie (v1.3.1)

While specific MVB12A expression data across all chicken tissues isn't comprehensively documented in the provided search results, researchers can apply these established techniques to determine tissue-specific expression patterns.

How should I design experiments to study MVB12A function in chicken cell lines?

A comprehensive experimental design to study MVB12A function should include:

• Loss-of-function approaches:

  • CRISPR/Cas9-mediated knockout of MVB12A in chicken cell lines (DF-1 or primary cells)

  • siRNA-mediated knockdown for transient suppression

  • Design guide RNAs targeting conserved exons of MVB12A

• Gain-of-function approaches:

  • Overexpression of wildtype MVB12A

  • Domain-specific mutants to identify functional regions

  • Tagged versions (GFP or FLAG) for localization studies

• Functional assays:

  • Measure endocytosis rates using fluorescently labeled transferrin

  • Analyze EGFR degradation kinetics using western blotting

  • Assess viral budding efficiency in infected cells

  • Examine intracellular protein trafficking using confocal microscopy

• Experimental controls:

  • Include both positive controls (known ESCRT component manipulation)

  • Negative controls (non-targeting guide RNAs or scrambled siRNAs)

  • Rescue experiments with wild-type MVB12A to confirm specificity

• Statistical analysis:

  • Apply factorial design analysis for multivariable experiments

  • Use ANOVA for assessing significant differences between treatment groups

  • Include at least 3 biological replicates per condition for statistical power

What are the optimal conditions for expressing recombinant chicken MVB12A in bacteria or other expression systems?

For bacterial expression of recombinant chicken MVB12A, implement the following methodological approach:

Bacterial Expression System:

• Clone the chicken MVB12A coding sequence into pET or pGEX vectors for E. coli expression

• Transform into expression strains (BL21(DE3), Rosetta, or Origami for disulfide bonds)

• Optimize induction conditions:

  • IPTG concentration: Test 0.1 mM, 0.5 mM, and 1.0 mM

  • Temperature: Compare 16°C, 25°C, and 37°C (lower temperatures often improve solubility)

  • Induction time: Test 4h, 8h, and overnight induction

• Include solubility tags if needed (SUMO, MBP, or GST)

Mammalian Cell Expression:

• Clone MVB12A into mammalian expression vectors with strong promoters (CMV)

• Transfect into HEK293 or CHO cells using lipofection or electroporation

• Create stable cell lines using selection markers

Alternative System: Chicken Oviduct Expression
Consider using the chicken itself as an expression system, as chickens have been successfully used as bioreactors for recombinant protein production:

• Design a construct with:

  • Oviduct-specific promoter (like ovalbumin promoter)

  • MVB12A coding sequence

  • Appropriate purification tags

• Generate transgenic chickens using CRISPR/Cas9 technology

• Harvest recombinant protein from egg whites

This approach has proved successful for other recombinant proteins in chickens, with expression levels reaching multiple mg/mL in egg whites and proper post-translational modifications .

What statistical design should I use for analyzing MVB12A expression changes across different chicken breeds?

For analyzing MVB12A expression differences across chicken breeds, implement a robust statistical design:

• Experimental Design Options:

  • Completely Randomized Design: For comparing MVB12A expression across multiple breeds

  • Randomized Block Design: When controlling for environmental factors

  • Factorial Design: When analyzing interactions between breed and environmental factors

• Sample Size Determination:

  • Perform power analysis before experimentation

  • Aim for at least 8 biological replicates per breed (based on similar gene expression studies)

  • Consider nested design if analyzing multiple tissues within each breed

• Statistical Analysis Methods:

  • One-way ANOVA for single-factor comparisons

  • Two-way ANOVA for analyzing breed and environmental interactions

  • Linear mixed models for handling random effects

  • Calculate variance components to assess within-breed vs. between-breed variation

• Post-hoc Analysis:

  • Tukey's HSD or Bonferroni correction for multiple comparisons

  • FDR (False Discovery Rate) control when analyzing genome-wide expression

• Presentation of Results:

  • Use box plots showing median, quartiles, and outliers

  • Include dot plots of individual data points for transparency

  • Provide effect sizes alongside p-values

For analysis of expression data across breeds, specialized software like R with packages (limma, edgeR) or dedicated experimental design software like CycDesigN can be utilized to generate and analyze appropriate designs .

How does chicken MVB12A interact with other components of the ESCRT-I complex and what are the structural implications?

The interaction between chicken MVB12A and other ESCRT-I components involves complex structural relationships:

Interaction Analysis Methodology:

• Structural characterization:

  • X-ray crystallography or Cryo-EM to determine the 3D structure

  • Focus on the dynamic helical domain that connects the GLUE domain to the rest of the ESCRT-II core

• Binding domain identification:

  • Use yeast two-hybrid or pull-down assays to map interaction domains

  • Create truncated constructs to identify minimal binding regions

  • Apply site-directed mutagenesis to identify critical residues

• In vivo verification:

  • Co-immunoprecipitation from chicken cell lysates

  • FRET or BiFC analysis for real-time interaction studies

  • Cross-linking mass spectrometry to capture transient interactions

MVB12A likely interacts with VPS28 through a specific helix, based on structural data from human ESCRT-II complexes, which reveal that "ESCRT-II binds to the ESCRT-I VPS28 C-terminal domain subunit through a helix immediately C-terminal to the VPS36-GLUE domain" .

The complex has three lobes containing VPS22, VPS36, and two copies of VPS25, forming a dynamic structure. MVB12A's interaction with these components affects membrane targeting through both the GLUE domain and the N-terminal regions .

What role does MVB12A play in chicken embryonic development and how can I investigate developmental timing of expression?

Role in Embryonic Development:
MVB12A likely plays important roles in chicken embryonic development through its functions in vesicular trafficking and receptor signaling. While specific studies on MVB12A in chicken embryogenesis aren't detailed in the provided search results, investigating its role requires:

Methodological Approach:

• Temporal expression profiling:

  • Collect embryos at key developmental stages (HH stages 1-45)

  • Perform RNA-seq and proteomics at each stage

  • Create an expression timeline of MVB12A throughout development

• Spatial expression analysis:

  • In situ hybridization to visualize MVB12A expression patterns

  • Immunohistochemistry with MVB12A-specific antibodies

  • Tissue-specific RT-PCR for quantitative comparison

• Functional studies:

  • CRISPR/Cas9-mediated knockout in chicken embryos

  • Morpholino-based knockdown for stage-specific inhibition

  • Ex ovo culturing for live imaging of manipulated embryos

• Signaling pathway investigation:

  • Analyze interaction with known developmental pathways (Notch, Wnt, FGF)

  • Look for co-expression with pathway components like those in the Notch signaling pathway, which has been shown to regulate adult neural stem cell maintenance through genes like Sox2

This experimental approach allows for comprehensive characterization of MVB12A's developmental roles and potential tissue-specific functions during embryogenesis.

How can CRISPR/Cas9 technology be optimized for generating MVB12A knock-in chickens for recombinant protein production?

Optimized CRISPR/Cas9 Protocol for MVB12A Knock-in Chickens:

• Target Selection and Guide RNA Design:

  • Identify optimal genomic loci for MVB12A insertion (ovalbumin locus is preferred)

  • Design multiple guide RNAs using predictive algorithms that minimize off-target effects

  • Test guide RNA efficiency in chicken cell lines before in vivo application

• Donor Template Construction:

  • Design homology arms (≥800 bp each) flanking the cut site

  • Include the MVB12A coding sequence with appropriate regulatory elements

  • Add selectable markers for PGC screening (e.g., puromycin resistance)

  • Consider adding a purification tag for easier protein isolation

• Delivery to Primordial Germ Cells (PGCs):

  • Isolate chicken PGCs from embryonic blood at stage HH14-17

  • Transfect PGCs with CRISPR/Cas9 components using Lipofectamine 2000 (2 μg donor plasmid with 2 μg CRISPR/Cas9 expression plasmid)

  • Select transfected cells with 1 μg/ml puromycin for 24 hours

  • Verify knock-in using PCR and sequencing

• PGC Transplantation and Chicken Generation:

  • Inject modified PGCs into recipient embryos

  • Raise chimeric chickens to sexual maturity

  • Screen offspring for germline transmission using PCR

• Protein Expression Analysis:

  • Collect eggs from G1 hens

  • Analyze egg white for MVB12A expression using ELISA and Western blotting

  • Assess protein functionality through appropriate bioassays

This methodology is based on successful approaches used for other recombinant proteins in chickens, such as human adiponectin, which achieved expression levels of 1.47-4.59 mg/mL in egg whites .

How can I analyze potential post-translational modifications of chicken MVB12A and their functional significance?

Comprehensive PTM Analysis Protocol:

• Protein Purification Strategy:

  • Express tagged MVB12A in chicken cells or recombinant systems

  • Implement two-step purification (e.g., affinity chromatography followed by size exclusion)

  • Maintain native conditions to preserve modifications

• Mass Spectrometry Workflow:

  • Perform tryptic digestion with complementary proteases for complete coverage

  • Apply both bottom-up (peptide) and top-down (intact protein) MS approaches

  • Use multiple fragmentation methods (CID, ETD, HCD) for comprehensive PTM identification

  • Implement specialized enrichment strategies for specific modifications:

    • Phosphorylation: TiO₂ or IMAC enrichment

    • Glycosylation: Lectin affinity or hydrazide chemistry

    • Ubiquitination: K-ε-GG antibody enrichment

• Bioinformatic Analysis:

  • Use multiple search engines (Mascot, SEQUEST, MaxQuant) with appropriate PTM settings

  • Apply false discovery rate control at both peptide and protein levels

  • Quantify modification stoichiometry using label-free or labeled approaches

  • Map modifications to protein structure using available structural data

• Functional Validation:

  • Generate site-specific mutants (e.g., phospho-null S/T→A or phospho-mimetic S/T→D/E)

  • Compare wildtype and mutant proteins in functional assays

  • Assess interaction changes using co-IP or proximity labeling

• Biological Context Analysis:

  • Compare modifications across tissues and developmental stages

  • Identify regulatory enzymes (kinases, glycosyltransferases) responsible for each modification

  • Determine conservation of modification sites across species

This methodological approach would provide comprehensive insights into the PTM landscape of chicken MVB12A and its functional relevance.

How can studying chicken MVB12A contribute to our understanding of ESCRT complex evolution across species?

Evolutionary analysis of chicken MVB12A can provide significant insights through these methodological approaches:

• Phylogenetic Analysis Framework:

  • Obtain MVB12A sequences from diverse species (mammals, birds, reptiles, amphibians, fish)

  • Align sequences using MUSCLE or MAFFT algorithms

  • Construct phylogenetic trees using Maximum Likelihood or Bayesian approaches

  • Calculate evolutionary rates across different lineages

  • Identify regions under positive or purifying selection

• Structural Conservation Analysis:

  • Compare crystal structures across species when available

  • Predict structures using AlphaFold2 for species lacking experimental structures

  • Identify conserved interaction interfaces with other ESCRT components

  • Map conservation scores onto 3D structures to visualize evolutionary constraints

• Functional Domain Comparison:

  • The ESCRT-II complex has a conserved structure with three lobes containing specific subunits

  • Analyze how the "dynamic helical domain to which both the VPS22 and VPS36 subunits contribute" evolved

  • Compare membrane-binding mechanisms across species, noting that "ESCRT-II is targeted to endosomal membranes by the lipid binding activities of both the Vps36 GLUE domain and the first helix of Vps22"

• Experimental Validation:

  • Perform cross-species complementation studies

  • Test if chicken MVB12A can rescue defects in MVB12A-deficient cells from other species

  • Compare interaction networks using cross-species pull-down assays

This approach would provide insights into how the ESCRT machinery evolved across vertebrates and how chicken-specific adaptations might relate to avian-specific cellular processes.

What are the most effective techniques for visualizing MVB12A localization and trafficking in chicken cells?

To effectively visualize MVB12A localization and trafficking in chicken cells, implement the following advanced imaging techniques:

Methodological Approach:

• Fluorescent Protein Fusion Strategies:

  • Create C-terminal and N-terminal fusions with monomeric fluorescent proteins

  • Test multiple fluorophores (mNeonGreen, mScarlet, HaloTag) to ensure functionality

  • Generate stable chicken cell lines expressing fusion proteins at near-endogenous levels

  • Validate localization with antibody staining of endogenous protein

• Live Cell Imaging Setup:

  • Use spinning disk confocal microscopy for rapid acquisition with minimal photobleaching

  • Implement temperature and CO₂ control for long-term imaging

  • Apply deconvolution algorithms to improve signal-to-noise ratio

  • Optimize acquisition parameters (exposure time, laser power) to minimize phototoxicity

• Advanced Visualization Techniques:

  • FRAP (Fluorescence Recovery After Photobleaching) to measure protein dynamics

  • Implement photo-convertible fluorophores to track specific protein populations

  • Apply TIRF microscopy to visualize membrane-proximal events

  • Use lattice light-sheet microscopy for high-resolution 3D tracking with reduced phototoxicity

• Colocalization Studies:

  • Standard markers: Rab5 (early endosomes), Rab7 (late endosomes), LAMP1 (lysosomes)

  • ESCRT components: VPS28, VPS22, VPS25, VPS36

  • Implement proper controls for spectral bleed-through

  • Use quantitative colocalization metrics (Pearson's, Manders' coefficients)

• Quantitative Analysis Workflow:

  • Track vesicle movement using automated particle tracking

  • Measure parameters like velocity, displacement, and directional persistence

  • Apply diffusion analysis to distinguish between random and directed movement

  • Use machine learning algorithms for classification of trafficking behaviors

This comprehensive approach provides both qualitative and quantitative data on MVB12A dynamics in chicken cells.

How can I investigate the potential role of MVB12A in chicken immune response and viral infection?

To investigate MVB12A's role in chicken immune response and viral infection, implement this systematic research approach:

Experimental Framework:

• Expression Analysis in Immune Challenge:

  • Challenge chicken cells or tissues with PAMPs (LPS, poly(I:C), CpG DNA)

  • Measure MVB12A expression changes using qRT-PCR and Western blotting

  • Analyze expression in different immune cell populations (macrophages, dendritic cells, T cells)

  • Compare response kinetics with known immune response genes

• Viral Infection Models:

  • Utilize relevant avian viruses (avian influenza, Newcastle disease virus, infectious bronchitis virus)

  • Create MVB12A knockdown and overexpression systems in chicken cell lines

  • Measure viral replication efficiency through plaque assays and qPCR

  • Assess changes in virus budding and release using electron microscopy

• Interaction Studies:

  • Identify potential interactions with viral proteins using:

    • Yeast two-hybrid screening

    • Co-immunoprecipitation followed by mass spectrometry

    • Bimolecular fluorescence complementation (BiFC)

  • Validate interactions with recombinant protein pull-downs

• Functional Assays:

  • Measure changes in cytokine production (IFN-α/β, IL-1β, IL-6) in MVB12A-modified cells

  • Assess antigen presentation efficiency in antigen-presenting cells

  • Evaluate the impact on MHC-I and MHC-II surface expression

  • Test antibody production in vivo using MVB12A-modified chicken models

• Mechanistic Investigation:

  • Analyze the role of MVB12A in exosome production during immune responses

  • Investigate autophagy regulation in response to infection

  • Examine impact on signaling pathways (JAK/STAT, NF-κB) during immune activation

This approach would comprehensively characterize the immunological functions of MVB12A in chickens and its relevance to viral pathogenesis and host defense.

How can recombinant chicken MVB12A be utilized in studying membrane trafficking disorders in avian and mammalian systems?

Recombinant chicken MVB12A can serve as a valuable tool for investigating membrane trafficking disorders through these methodological applications:

• Comparative Model System Development:

  • Express chicken MVB12A in mammalian cell lines with trafficking defects

  • Compare rescue efficiency with mammalian orthologs

  • Create chimeric proteins with domain swaps to identify functional conservation

  • Develop fluorescently tagged versions for real-time tracking in different cellular backgrounds

• Therapeutic Screening Platform:

  • Use MVB12A-dependent trafficking assays to screen compound libraries

  • Establish quantitative readouts (receptor degradation, cargo sorting)

  • Apply to disorders involving:

    • Lysosomal storage diseases

    • Neurodegenerative conditions with trafficking defects

    • Viral budding-related pathologies

• Structural Studies for Rational Drug Design:

  • Leverage the compact structure of ESCRT-II, which "has three lobes and contains one copy each of VPS22 and VPS36, and two copies of VPS25"

  • Target the "dynamic helical domain to which both the VPS22 and VPS36 subunits contribute"

  • Develop small molecules that modulate MVB12A interactions with other ESCRT components

• Biomarker Development:

  • Identify MVB12A modifications or expression changes in disease states

  • Develop antibodies specific to modified forms

  • Create diagnostic assays for trafficking-related disorders

This translational approach bridges basic research on MVB12A structure-function with potential clinical applications in trafficking disorders.

What are the experimental considerations for using engineered chicken cell lines to produce recombinant MVB12A for structural studies?

For optimal production of recombinant MVB12A in engineered chicken cell lines for structural studies, consider these methodological details:

Production System Optimization:

• Cell Line Selection and Engineering:

  • Compare DF-1 (immortalized chicken fibroblasts) vs. HD11 (macrophage-like) cell lines

  • Develop stable cell lines using lentiviral transduction or CRISPR knock-in

  • Include inducible promoter systems (Tet-On/Off) for controlled expression

  • Engineer cell lines to minimize proteolytic degradation (knockout relevant proteases)

• Expression Vector Design:

  • Optimize codon usage for chicken cell expression

  • Include purification tags compatible with structural studies (His6, Twin-Strep)

  • Add cleavable linkers for tag removal (PreScission, TEV protease sites)

  • Consider fusion partners that enhance solubility while maintaining native structure

• Culture Conditions Optimization:

  • Perform DoE (Design of Experiments) to identify optimal parameters:

    • Temperature (37°C standard, 33°C for problematic proteins)

    • Media composition (serum levels, nutrient supplements)

    • Cell density at induction

    • Harvest timing

  • Implement factorial design experiments to identify parameter interactions

• Purification Strategy for Structural Integrity:

  • Develop gentle lysis procedures to maintain protein-protein interactions

  • Implement tandem affinity purification for high purity

  • Include size exclusion chromatography to ensure homogeneity

  • Verify structural integrity using circular dichroism before crystallization

• Quality Control Metrics:

  • Verify protein homogeneity by dynamic light scattering

  • Assess thermal stability using differential scanning fluorimetry

  • Confirm activity through functional assays

  • Validate native fold with limited proteolysis

This comprehensive approach ensures production of high-quality recombinant MVB12A suitable for crystallography, cryo-EM, or NMR studies.

How can I design experiments to investigate the potential of MVB12A as a biomarker for avian diseases?

To evaluate MVB12A's potential as an avian disease biomarker, implement this systematic experimental strategy:

Biomarker Validation Workflow:

• Expression Profiling in Disease Models:

  • Compare MVB12A expression across healthy and diseased tissues using:

    • qRT-PCR for mRNA quantification

    • Western blotting for protein levels

    • Immunohistochemistry for localization changes

  • Study multiple avian diseases:

    • Viral infections (avian influenza, Marek's disease)

    • Bacterial infections (colibacillosis, salmonellosis)

    • Metabolic disorders

• Biofluid Analysis:

  • Develop sensitive ELISA or other immunoassays for MVB12A detection

  • Compare MVB12A levels in:

    • Serum/plasma

    • Exosomes isolated from circulation

    • Other accessible biofluids

  • Correlate with disease progression and severity

• Diagnostic Test Development:

  • Design statistical experiments to determine:

    • Sensitivity and specificity

    • Positive and negative predictive values

    • Receiver operating characteristic (ROC) curves

  • Compare performance against existing diagnostic methods

  • Validate in blinded samples from diverse populations

• Clinical Correlation Studies:

  • Design prospective studies following DOE principles:

    • Factorial design with multiple variables (age, breed, disease state)

    • Calculate appropriate sample sizes for statistical power

    • Include relevant controls and standards

  • Implement statistical analysis methods as described in comprehensive research design texts

• MVB12A Isoform and Modification Analysis:

  • Identify disease-specific PTMs or splice variants

  • Develop assays specific to modified forms

  • Correlate modifications with disease mechanisms