Recombinant Chicken Ashwin (RCJMB04_12m8)

What is Recombinant Chicken Ashwin (RCJMB04_12m8)?

Recombinant Chicken Ashwin (RCJMB04_12m8) is a full-length protein derived from Gallus gallus (chicken) with UniProt accession number Q5ZK74. It is typically expressed in E.coli expression systems and is supplied with purity greater than 85% as determined by SDS-PAGE analysis. The protein consists of 241 amino acids and is also known simply as Ashwin .

What are the optimal storage and handling conditions for Recombinant Chicken Ashwin?

For optimal stability and experimental reproducibility, researchers should adhere to the following storage guidelines:

Repeated freezing and thawing cycles should be avoided as they can compromise protein integrity. Working aliquots may be stored at 4°C for up to one week. Prior to reconstitution, the vial should be briefly centrifuged to bring contents to the bottom .

How should Recombinant Chicken Ashwin be reconstituted for experimental use?

The recommended reconstitution protocol is as follows:

• Centrifuge the vial briefly to collect all material at the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (standard is 50%)

• Prepare small working aliquots to minimize freeze-thaw cycles

• Store reconstituted aliquots at -20°C/-80°C for long-term storage

This protocol helps maintain protein stability and activity for downstream applications .

How can Recombinant Chicken Ashwin be incorporated into experimental designs for avian virus research?

When designing experiments to investigate potential roles of Recombinant Chicken Ashwin in avian virus research, researchers should consider the following methodological approach:

• Establish experimental groups: Include treatment groups receiving Recombinant Chicken Ashwin at different concentrations and appropriate control groups

• Consider timing variables: Design both preventive (pre-infection) and therapeutic (post-infection) administration protocols

• Account for age-dependent effects: Test responses in different age groups (e.g., 7-day-old and 33-day-old SPF chickens), as age-dependent responses have been observed with other recombinant chicken proteins

• Measure relevant endpoints: Quantify viral load in target tissues (trachea, lungs), assess clinical signs, and monitor survival rates

• Analyze gene expression changes: Examine whether Ashwin treatment modulates expression of IFN-stimulated genes (ISGs) such as 2',5'-oligoadenylate synthetase and Mx1

This approach adapts methodologies that have proven successful with other recombinant chicken proteins such as chicken interferon-alpha (ChIFN-α) in avian influenza virus research .

What experimental methods are recommended for investigating potential interactions between Recombinant Chicken Ashwin and avian influenza virus proteins?

A comprehensive investigation of potential interactions should employ multiple complementary techniques:

• In vitro binding assays: Utilize co-immunoprecipitation, ELISA, or surface plasmon resonance to detect direct interactions between Recombinant Chicken Ashwin and viral proteins

• Functional interference studies: Assess whether pre-incubation of virus with Recombinant Chicken Ashwin affects viral entry or replication in cell culture systems

• Confocal microscopy: Examine co-localization of fluorescently-labeled Recombinant Chicken Ashwin with viral components during different stages of infection

• Cross-linking experiments: Identify potential transient interactions through chemical cross-linking followed by mass spectrometry

• Recombinant virus systems: Generate recombinant viruses with reporter genes to quantify how Ashwin affects viral replication stages

These methods can help determine whether Ashwin plays a direct role in avian host defense mechanisms against viral pathogens, similar to roles observed with other chicken proteins in influenza virus studies .

How can researchers resolve data conflicts in experiments involving Recombinant Chicken Ashwin?

When encountering conflicting experimental results with Recombinant Chicken Ashwin, researchers should implement a systematic approach to data reconciliation:

• Source reliability estimation: Develop an optimization framework that models the reliability of different data sources, assigning appropriate weights to each source based on consistency and reproducibility

• Heterogeneous data integration: Apply specialized loss functions for different data types (continuous measurements, categorical observations, sequence data)

• Normalization assessment: Carefully evaluate whether methodological differences in normalization could be introducing false positives or negatives (e.g., failure to normalize for differences in library size when comparing gene expression data)

• Iterative truth discovery: Implement algorithms that iteratively update both the estimated "ground truth" and source weights until convergence

• Strategic experimental design: Design critical experiments specifically to address points of contradiction between existing datasets

This approach adapts methods from truth discovery and conflict resolution in heterogeneous data that have proven effective in resolving contradictions in complex biological datasets .

What microarray analysis strategies are recommended for studying Recombinant Chicken Ashwin's effects on host gene expression?

For robust microarray analysis of Ashwin's effects on host gene expression, researchers should follow this methodological framework:

• Experimental design:

  • Include appropriate biological replicates (minimum n=3 per condition)

  • Collect samples at multiple time points post-treatment

  • Include relevant control groups (untreated, vehicle-treated, irrelevant protein treatment)

• Technical implementation:

  • Extract RNA using methods that preserve integrity (RIN > 8.0)

  • Perform quality control at each step (RNA quality, labeling efficiency, hybridization quality)

  • Use appropriate normalization methods for the specific microarray platform

• Data analysis:

  • Apply statistical tests with multiple testing correction (FDR < 0.01)

  • Identify differentially expressed genes (fold change > 1.5)

  • Perform pathway and gene ontology enrichment analysis

  • Validate key findings using quantitative RT-PCR

• Integration with survival data:

  • Correlate gene expression patterns with phenotypic outcomes

  • Identify gene signatures that predict survivability following challenges

Following this approach has successfully identified genes whose expression correlates with survivability in chickens infected with recombinant viruses possessing H5N1 surface antigens, including CD274, RNF19B, OASL, ZC3HAV1, PLA2G6, GCH1, and USP18 (P < 0.01) .

How can CRISPR/Cas9 techniques be applied to study Recombinant Chicken Ashwin function in vivo?

CRISPR/Cas9 genome editing offers powerful approaches to investigate Ashwin function in chickens:

• Target identification:

  • Perform in silico analysis to identify conserved domains within the Ashwin gene

  • Design guide RNAs targeting critical regions (e.g., binding domains, active sites)

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

• Genetic modification strategies:

  • Gene knockout: Complete deletion or frameshift mutations in the Ashwin gene

  • Point mutations: Introduction of specific amino acid changes to test functional hypotheses

  • Regulatory modifications: Alterations to promoter or enhancer regions to modulate expression

• Delivery methods for chicken applications:

  • Primordial germ cell modification followed by germline transmission

  • Direct injection into embryos at appropriate developmental stages

  • Lentiviral vector delivery systems

• Phenotypic characterization:

  • Challenge studies with viral pathogens

  • Comprehensive immunological profiling

  • Transcriptomic analysis of modified versus wild-type chickens

This approach adapts successful CRISPR/Cas9 strategies used to generate ALV-J resistant chickens through modification of the chicken Na+/H+ exchanger type 1 (chNHE1) .

What are best practices for experimental design when studying potential roles of Recombinant Chicken Ashwin in host-pathogen interactions?

A robust experimental design for investigating Ashwin's role in host-pathogen interactions should include:

• Clearly defined research question:

  • Formulate a specific hypothesis (e.g., "How does Recombinant Chicken Ashwin treatment affect viral replication in chicken cells?")

  • Identify precise independent variables (Ashwin concentration, timing of treatment) and dependent variables (viral load, gene expression)

• Variable control:

  • Independent variable: Recombinant Chicken Ashwin concentration (e.g., 0, 10, 50, 100 ng/mL)

  • Dependent variable: Measure with appropriate units and precision (viral copies/mL, fold change in gene expression)

  • Controlled variables: Maintain consistent temperature, pH, cell density, etc.

  • Constants: Use same viral strain, cell type, and detection methods across experiments

• Detailed protocol development:

  • Include at least three trials for each experimental condition

  • Document procedures with sufficient detail for reproduction

  • Create labeled diagrams of experimental setups

  • Include appropriate positive and negative controls

• Data analysis plan:

  • Select appropriate statistical tests based on data distribution

  • Determine sample sizes using power analysis

  • Plan for data normalization and transformation if needed

  • Establish clear criteria for interpreting results

Following these principles will strengthen experimental rigor and reproducibility, as emphasized in established experimental design frameworks .

How can researchers investigate potential interactions between Recombinant Chicken Ashwin and host immunity during avian influenza infection?

To investigate Ashwin's interactions with host immunity, researchers should employ a multifaceted approach:

• In vitro immune cell assays:

  • Isolate primary chicken immune cells (macrophages, heterophils, lymphocytes)

  • Measure changes in cellular activation markers following Ashwin treatment

  • Assess cytokine/chemokine production using ELISA or multiplex assays

  • Evaluate changes in innate immune signaling pathways (NF-κB, IRF3/7)

• Ex vivo tissue explants:

  • Treat chicken tracheal or lung explants with Recombinant Chicken Ashwin

  • Challenge with avian influenza virus

  • Measure local immune responses and viral replication

• Transcriptomic profiling:

  • Compare gene expression patterns in immune-relevant tissues with and without Ashwin treatment

  • Focus on interferon-stimulated genes and pattern recognition receptors

  • Validate key findings using quantitative RT-PCR and protein detection methods

• In vivo challenge studies:

  • Administer Recombinant Chicken Ashwin via different routes (oral, intranasal, intravenous)

  • Challenge with avian influenza virus

  • Monitor clinical signs, viral shedding, and survival

  • Analyze T-dependent lymphocyte responses in different age groups

This approach synthesizes methods used to study chicken interferon-alpha and recombinant viruses possessing H5N1 surface antigens, adapting them to investigate Ashwin's potential immunomodulatory functions .

What approaches can be used to evaluate data reproducibility in Recombinant Chicken Ashwin research?

To ensure robust, reproducible findings in Ashwin research, implement these methodological approaches:

• Independent verification:

  • Repeat key experiments in different laboratories

  • Use different batches of Recombinant Chicken Ashwin

  • Employ alternative methodologies to confirm findings

• Systematic review of potential artifacts:

  • Check for library size normalization errors in sequencing data

  • Verify proper controls for batch effects

  • Examine methodological inconsistencies between datasets

  • Evaluate whether appropriate statistical tests were applied

• Dataset integration strategies:

  • Use meta-analysis approaches to combine multiple experimental results

  • Apply correction factors for cross-laboratory or cross-platform variations

  • Implement statistical methods that account for heterogeneity between studies

• Documentation of methodological details:

  • Provide comprehensive information about Recombinant Chicken Ashwin source, purity, and preparation

  • Document exact experimental conditions, including buffer compositions

  • Report all statistical analyses performed, including unsuccessful approaches

This framework adapts strategies identified in analyses of irreproducible results in genomic studies, where failure to normalize for differences in sample preparation led to false positive findings .