Recombinant Chicken Actin-related protein 5 (ACTR5), partial

What is the structural composition of chicken ACTR5?

ACTR5 belongs to the family of actin-related proteins with high sequence similarity to actin. The protein consists of N-domain and C-domain that are conserved among conventional actins and other ARPs, plus three unique domains: N-terminal (S1), central (S2), and C-terminal (S3) regions . Unlike conventional actins, ACTR5 has specific structural features that enable its nuclear localization and interaction with various transcription factors, making it functionally distinct from cytoplasmic actin .

How is ACTR5 expression regulated in chicken tissues?

ACTR5 expression varies significantly across chicken tissues and developmental stages. RNA-seq data analysis shows that ACTR5 levels in adult tissues are reduced to approximately half of that observed in embryonic tissues . Expression is particularly low in skeletal muscle, heart, and aorta, suggesting tissue-specific regulation . In pathological conditions such as cardiomyopathy, ACTR5 expression increases, indicating its potential role in disease processes . Regulation may occur through alternative splicing coupled to nonsense-mediated mRNA decay (AS-NMD), which contributes to post-transcriptional fine-tuning of gene expression .

What are the recommended approaches for cloning and expressing recombinant chicken ACTR5?

For effective cloning and expression of chicken ACTR5, researchers should:

• Design gene-specific primers based on the chicken ACTR5 sequence (similar to the approach used for ADRA2A in chicken studies)

• Amplify the complete open reading frame (ORF) from appropriate chicken tissue (such as brain or embryonic tissue)

• Clone the amplified PCR products into an expression vector such as pcDNA3.1(+)

• For recombinant protein production, insect cell culture systems have proven effective for producing functional recombinant proteins from chicken

• For in vivo expression studies, viral vector systems such as RCAS(A) retrovirus can be used for targeted expression in chicken embryos

How can researchers effectively evaluate ACTR5 protein-protein interactions in chicken tissues?

To study ACTR5 interactions with other proteins:

• Co-immunoprecipitation (Co-IP) assays using anti-ACTR5 antibodies with chicken tissue lysates can identify endogenous interaction partners

• Recombinant ACTR5 protein can be used in pull-down assays with brain or other tissue lysates to identify tissue-specific interacting proteins

• For transcription factor interactions, chromatin immunoprecipitation (ChIP) assays can determine ACTR5 occupancy at specific genomic loci

• Reporter assays using chicken cell lines (such as DF-1) can assess the functional impact of ACTR5 on transcriptional regulation

What viral vector systems are most suitable for expressing recombinant ACTR5 in chicken models?

Several viral vector systems have proven effective for chicken studies:

• RCAS(A) (Replication-Competent Avian Sarcoma/Leukosis virus type A): Particularly useful for embryonic studies, this system allows for targeted gene transfer to proliferative cells without requiring tissue-specific promoters

• Lentiviral vectors based on Equine Infectious Anemia Virus (EIAV): Suitable for producing genetically modified chickens with stable transgene integration and inheritance

• Adeno-associated virus serotype 6 (AAV6): Shows high transduction efficiency in chicken heart tissue and has been successfully used to overexpress proteins in chicken tissues

For embryonic studies, microinjection of RCAS(A) retrovirus into the embryonic chicken lens or other target tissues provides an efficient method for studying in situ expression and function of proteins during development .

How does ACTR5 regulate myogenic differentiation in chicken cells?

ACTR5 functions as an inhibitory regulator of myogenic differentiation in chicken:

• ACTR5 inhibits myogenic gene expression by binding to and interfering with the function of myogenic regulatory factors (MRFs)

• In chicken muscle cells, ACTR5 expression decreases during differentiation, suggesting its downregulation is necessary for proper muscle development

• Mechanistically, ACTR5 competes with other transcription factors for binding to MRFs, preventing their activation of target genes

• Overexpression of ACTR5 in chicken muscle cells inhibits the expression of muscle-specific genes and reduces myotube formation

For researchers studying muscle development in chickens, modulating ACTR5 levels through recombinant protein expression or gene knockdown approaches can provide insights into the regulation of myogenic differentiation.

What role does ACTR5 play in chicken heart development and cardiac gene expression?

ACTR5 acts as a suppressor of cardiac gene expression through multiple mechanisms:

• It binds to the N-terminus of cardiac MYOCD (myocardin), a critical transcription factor for cardiac development

• ACTR5 competes with MEF2 (myocyte enhancer factor 2) to interact with cardiac MYOCD and suppresses MYOCD-MEF2-mediated transactivation of cardiac genes

• Exogenous overexpression of ACTR5 using AAV6 vectors in chicken hearts induces cardiac hypertrophy and fibrosis with decreased expression of SRF- and MEF2-regulated cardiac genes

• RNA-seq analysis has identified 650 genes that are repressed by ACTR5 in cardiac tissue

The expression table below summarizes ACTR5 expression patterns in cardiac tissue:

How can ACTR5 be utilized in studying chicken primordial germ cell (PGC) development?

ACTR5 can be used as a tool in PGC research contexts:

• Studying interactions between ACTR5 and developmental regulators like DDX5, which is upregulated during chicken PGC formation

• Recombinant ACTR5 can be employed in biochemical assays to understand protein interactions in the regulatory networks controlling PGC specification

• In combination with gene editing technologies, ACTR5 manipulation can help elucidate chromatin remodeling mechanisms during PGC development

• ACTR5 expression analysis can serve as a molecular marker for specific developmental stages of chicken PGCs

What are the optimal expression systems for producing recombinant chicken ACTR5?

Several expression systems have been evaluated for chicken protein production:

• Insect cell culture systems: Offer proper post-translational modifications and have been successfully used for producing functional recombinant chicken proteins

• Chicken DF-1 cell line: A continuous cell line derived from chicken embryo fibroblasts that can be used for stable expression of recombinant chicken proteins

• Egg bioreactor systems: Genetic modification of chickens allows for the production of recombinant proteins in eggs, providing a scalable system for protein production

For ACTR5 specifically, researchers should consider:

• Using the EF1α promoter for continuous expression in chicken cell lines

• Adding appropriate purification tags (His, FLAG, etc.) without disrupting functional domains

• Optimizing codon usage for the expression system to improve yield

What purification strategies are most effective for recombinant chicken ACTR5?

For optimal purification of recombinant chicken ACTR5:

• Expression with affinity tags (His, GST, or FLAG) facilitates single-step purification

• For structural and functional studies, consider removing tags through specific protease cleavage sites

• Size exclusion chromatography can be used as a final polishing step to ensure high purity

• When working with chicken INO80 complex components, co-expression with interacting partners like Ies6 may improve stability and solubility

• Buffer optimization is critical, with typical conditions including 20-50 mM Tris-HCl (pH 7.5-8.0), 150-300 mM NaCl, 1-5 mM DTT, and 5-10% glycerol

How can the functionality of recombinant chicken ACTR5 be validated?

To validate the functionality of purified recombinant ACTR5:

• Binding assays: Verify interactions with known partners such as transcription factors (MYOCD, MyoD) using pull-down assays or surface plasmon resonance

• Chromatin remodeling assays: Test the ability of ACTR5 to participate in nucleosome remodeling when incorporated into the INO80 complex

• Cell-based reporter assays: Evaluate the effect of recombinant ACTR5 on promoter activity of target genes (e.g., MyoG promoter)

• In vitro competition assays: Determine if recombinant ACTR5 can compete with other factors for binding to transcription factors

How do you address potential off-target effects when studying ACTR5 function in chicken models?

When investigating ACTR5 function in chicken models, researchers should implement multiple controls to address off-target effects:

• Use multiple independent siRNAs or shRNAs targeting different regions of ACTR5 to confirm phenotypes are due to specific knockdown

• Include rescue experiments with recombinant ACTR5 containing silent mutations that render it resistant to the knockdown approach

• For CRISPR-Cas9 genome editing, carefully validate edited sites through sequencing and analyze potential off-target modifications

• When using viral vectors for ACTR5 overexpression, include proper controls with empty vectors and unrelated proteins to account for effects of viral infection

• Consider tissue-specific or inducible expression systems to minimize developmental compensation mechanisms

What are the key considerations when interpreting contradictory data on ACTR5 function?

Contradictory findings regarding ACTR5 function may arise from:

• Context-dependent effects: ACTR5 functions differently in various tissues and developmental stages. For example, it shows distinct roles in cardiac versus skeletal muscle development

• INO80-dependent versus independent functions: Carefully determine whether phenotypes are due to ACTR5's role in the INO80 complex or its independent functions by parallel knockdown of other INO80 components

• Expression levels: Physiological versus overexpression conditions may yield different results. Use quantitative approaches to measure ACTR5 levels relative to endogenous expression

• Experimental systems: Results from in vitro cell culture may differ from in vivo findings. Validate key findings across multiple experimental systems

• Genetic background: Different chicken lines may show variable responses to ACTR5 manipulation, necessitating studies across diverse genetic backgrounds

How can researchers integrate ACTR5 studies with broader chromatin regulation research in avian models?

To place ACTR5 research within the broader context of chromatin regulation:

• Combine ACTR5 studies with genome-wide approaches like ChIP-seq and ATAC-seq to map chromatin accessibility changes

• Integrate transcriptomic data (RNA-seq) from ACTR5 manipulation experiments with existing datasets on chromatin state in chicken tissues

• Consider the evolutionary conservation of ACTR5 function by comparative studies across avian species and other vertebrates

• Investigate potential interactions between ACTR5-containing complexes and other chromatin modifiers specific to avian systems

• Develop chicken-specific antibodies and tools to better study native chromatin regulatory complexes in avian models

How can recombinant ACTR5 be utilized in vaccine development for avian diseases?

Recombinant ACTR5 may serve as a tool in vaccine research:

• As a fusion partner to enhance immunogenicity of subunit vaccines (similar to approaches used with other recombinant chicken proteins)

• In chimeric protein designs where ACTR5 domains are used to modulate immune responses

• For studying host-pathogen interactions in chicken cell lines and primary cells

• As part of adjuvant formulations to enhance vaccine efficacy in chicken models

Promising vaccine development approaches include:

• Combination of recombinant proteins with specific adjuvants has shown enhanced protection against high-dose viral challenges in chickens

• Passive immunization approaches using recombinant antibodies have demonstrated reduction in clinical disease and viral shedding

What role might ACTR5 play in chicken bioreactor systems for pharmaceutical protein production?

As research on chicken bioreactors advances, ACTR5 may contribute to this field:

• Modulation of ACTR5 could potentially enhance protein expression in chicken egg bioreactors by affecting chromatin accessibility at transgene loci

• Understanding ACTR5 regulation during oviduct development may inform better promoter design for egg white-specific expression of recombinant proteins

• ACTR5 knowledge could help identify ideal genomic loci for transgene insertion to maximize recombinant protein yield

• In cell-based evaluation systems for optimizing chicken bioreactors, ACTR5 manipulation might serve as a tool to predict in vivo expression patterns

Research has shown that knocking in promoters (like EF1α) at specific loci in chicken cells can achieve continuous expression of target proteins, suggesting potential approaches for stable transgene expression .

How does ACTR5 research contribute to understanding avian-specific aspects of gene regulation?

ACTR5 research provides insights into avian-specific gene regulation mechanisms:

• Studies of chicken ACTR5 interactions with transcription factors reveal evolutionary conservation and divergence in regulatory networks between avian and mammalian systems

• ACTR5's role in chromatin remodeling may explain avian-specific gene expression patterns during development

• Comparative analysis of ACTR5 function across species can illuminate the evolution of epigenetic regulation mechanisms

• Understanding tissue-specific expression patterns of ACTR5 in chickens may explain unique aspects of avian physiology and development