Recombinant Bovine Myogenin (MYOG)

What is Bovine Myogenin and how does it compare to human myogenic factors?

Bovine Myogenin (MYOG) belongs to the myogenic regulatory factor (MRF) family and shares significant homology with human myogenic factors. Research has identified a bovine cDNA, designated bmyf, that encodes a protein highly related to the human myogenic factor myf-5. Analysis reveals that human and bovine factors are 96% homologous in their predicted amino acid sequences. At the nucleotide level, bmyf and myf-5 are 92% identical in the coding region and 74% and 80% homologous in their 5'- and 3'-untranslated regions, respectively .

This high degree of conservation between species suggests critical functional importance and makes bovine myogenin an excellent model for comparative studies. Researchers should note that despite this homology, species-specific differences may affect experimental outcomes, particularly in cross-species applications.

What expression patterns does Bovine Myogenin exhibit in skeletal muscle tissue?

Bovine Myogenin exhibits tissue-specific expression patterns primarily localized to skeletal muscle. Studies have detected three distinct transcripts of approximately 1.5, 2, and 3 kb in bovine skeletal muscle tissue . The transcript diversity results from alternative polyadenylation, with the 1.5 kb transcript lacking sequences 3' to the polyadenylation signal at nucleotide 1415 in the bmyf cDNA, while both the 2 and 3 kb RNAs contain these sequences .

Temporal expression profiling shows that myogenin expression gradually increases during myogenesis, making it an excellent marker for muscle differentiation. This expression pattern is conserved across species, as similar patterns have been observed in mouse embryonic development where myogenin transcripts follow a defined spatial and temporal pattern during somite development .

What are the recommended approaches for cloning and expressing recombinant Bovine Myogenin?

To clone and express recombinant Bovine Myogenin, researchers should consider the following methodological approach:

• Primer Design: Design primers that specifically amplify the coding region of bovine myogenin. For example, when working with bmyf, researchers successfully amplified a fragment of 1000 bp from the last exon using primers designed with specific restriction sites (e.g., SalI and NotI) to facilitate subsequent cloning steps .

• cDNA Library Screening: Screen a bovine cDNA library from fetal skeletal muscle myoblasts using a probe to a conserved region. This approach has successfully identified positive recombinants containing the full coding sequence for bmyf .

• Expression Vector Construction: Subclone the amplified fragment into an appropriate expression vector. For optimal expression in mammalian cells, include a strong promoter suitable for muscle cell expression.

• Confirmation of Expression: Verify expression through Western blotting and functional assays, such as testing the ability of the recombinant protein to activate the myogenic program in C3H10T1/2 fibroblasts through stable and transient transfection experiments .

This methodology has proven successful in producing functional recombinant bovine myogenic factors that retain their biological activity.

How can researchers use CRISPR/Cas9 to study Myogenin function?

CRISPR/Cas9 technology offers powerful approaches for studying myogenin function through precise genome editing. Researchers have successfully employed the following methodology:

• sgRNA Design: Design a high-scoring sgRNA sequence targeting the desired region of the Myog gene. Tools such as the guide design tool from crispr.mit.edu (Zhang Lab) can help identify optimal targeting sequences .

• Targeting Construct Design: Create a recombination template consisting of homology arms corresponding to Myog sequences flanking the target site. Include reporter genes (e.g., tdTomato) preceded by a T2A peptide sequence to allow cleavage from the MYOG protein following translation, ensuring the native protein's function remains intact .

• Nuclear Localization: If studying protein localization, include a triple NLS sequence to ensure nuclear localization of fluorescent reporters .

• Verification Strategy: Design PCR primers that can distinguish between wild-type and edited alleles to verify successful genome modification .

This approach has been successfully used to generate reporter lines expressing fluorescent proteins under the control of the endogenous Myog promoter, allowing visualization of myogenin expression in live cells and enabling intravital microscopy studies of muscle differentiation dynamics .

How does Myogenin interact with other transcription factors in myogenic differentiation?

Myogenin participates in a complex regulatory network with other transcription factors during myogenic differentiation. Key interactions include:

• MyoD Coordination: Myogenin works in concert with MyoD, which functions as a master regulator of myogenic differentiation. MyoD can regulate myogenin expression, creating a feed-forward loop that reinforces the myogenic program .

• Ddx17 Interaction Network: Research has revealed that promoter-associated lncRNAs like Myoparr (expressed from the myogenin promoter region) interact with Ddx17, a transcriptional coactivator of MyoD. This interaction regulates the association between Ddx17 and the histone acetyltransferase PCAF, affecting chromatin accessibility and transcriptional activation of myogenic genes .

• Epigenetic Regulation: The myogenin promoter is subject to epigenetic regulation, including histone modifications that influence accessibility to transcription factors. These modifications are dynamically regulated during myogenic differentiation.

Understanding these interactions is crucial for researchers working with recombinant myogenin, as experimental designs should account for these regulatory networks when assessing myogenin function.

What role do promoter-associated lncRNAs play in Myogenin regulation?

Promoter-associated long non-coding RNAs (lncRNAs) have emerged as important regulators of myogenin expression. Key findings include:

• Myoparr Function: Myoparr is a promoter-derived lncRNA expressed from the promoter region of the myogenin gene in both mouse and human. It is essential for myoblast specification by activating neighboring myogenin expression and for myoblast cell cycle withdrawal by activating myogenic microRNA expression .

• Molecular Mechanism: Myoparr functions by binding to the myogenin promoter in cis and physically interacting with Ddx17, promoting the protein-protein interaction between Ddx17 and PCAF .

• Expression Correlation: The expression of Myoparr and myogenin is mutually correlated and regulated by MyoD and TGF-β. During myogenesis, Myoparr expression gradually increases alongside myogenin expression .

• Therapeutic Relevance: Myoparr also promotes skeletal muscle atrophy caused by denervation, and knockdown of Myoparr rescues muscle wasting in mice, suggesting potential therapeutic applications .

Researchers working with recombinant myogenin should consider the potential influence of these lncRNAs on experimental outcomes, particularly in systems where the native genomic context is altered.

How can recombinant Myogenin be used to study muscle fiber type composition?

Recombinant myogenin can serve as a valuable tool for investigating muscle fiber type composition through the following methodological approaches:

• Fiber Type Analysis: Studies have shown that myogenin expression influences the proportion of different muscle fiber types. Research with recombinant bovine somatotropin (rbST) demonstrated effects on myosin heavy chain expression patterns, with treated animals showing decreased proportions of MHC-I fibers and increased proportions of MHC-IIX fibers .

• Cross-sectional Area Measurement: When studying fiber type transitions, researchers should measure fiber cross-sectional area changes. As days on feed increased in one study, the area of MHC-I fibers decreased whereas MHC-IIA and IIX area increased .

• Immunohistochemical Analysis: Use immunohistochemistry with fiber type-specific antibodies to quantify changes in fiber type distribution following recombinant myogenin treatment or manipulation.

• Gene Expression Profiling: Combine recombinant myogenin treatments with gene expression analysis of fiber type-specific markers to determine transcriptional changes driving fiber type transitions.

This methodological approach provides insight into how myogenic regulatory factors like myogenin influence muscle phenotype, which has implications for both basic muscle biology and agricultural applications.

What methods are recommended for assessing the functional activity of recombinant Myogenin?

To assess the functional activity of recombinant Myogenin, researchers should employ a multi-faceted approach:

• Myogenic Conversion Assay: Determine whether the recombinant myogenin can activate the myogenic program in non-muscle cells such as C3H10T1/2 fibroblasts through stable and transient transfection experiments. Functional myogenin will induce expression of muscle-specific genes and morphological changes characteristic of myogenic differentiation .

• Gene Expression Analysis: Measure the expression of myogenin-dependent genes using RT-qPCR or RNA-sequencing to confirm transcriptional activation capabilities.

• Protein-Protein Interaction Studies: Assess the ability of recombinant myogenin to interact with known binding partners such as E-proteins and other transcription factors using co-immunoprecipitation or proximity ligation assays.

• DNA Binding Assays: Evaluate DNA binding activity using electrophoretic mobility shift assays (EMSA) or chromatin immunoprecipitation (ChIP) to confirm that the recombinant protein recognizes appropriate E-box elements.

• Reporter Gene Assays: Use reporter constructs containing myogenin-responsive elements to quantitatively measure transcriptional activation potential in various cell types.

These complementary approaches provide a comprehensive assessment of recombinant myogenin functionality across multiple dimensions of its biological activity.

What are the key considerations for designing in vivo experiments with recombinant Myogenin?

When designing in vivo experiments with recombinant Myogenin, researchers should consider the following methodological factors:

• Delivery Method: Choose appropriate delivery methods based on research objectives. For embryonic studies, consider electroporation of expression constructs into somites. For postnatal studies, viral vectors (AAV or lentivirus) or direct intramuscular injection may be more appropriate.

• Dosage and Timing: Carefully determine dosage and timing of administration. Myogenin expression is tightly regulated during development, and inappropriate timing or expression levels may lead to unintended phenotypes. Studies with rbST have demonstrated that timing of administration can significantly impact muscle biological activity .

• Animal Models: Select appropriate animal models. Mouse models offer genetic tractability, while bovine models provide direct relevance to cattle production. Consider using reporter models that allow visualization of myogenin activity, such as the MyogntdTom mouse line that exhibits robust reporter gene expression in myogenic cells .

• Ethical Considerations: Ensure protocols adhere to ethical guidelines. For example, animal handling should follow national and European Community guidelines with appropriate ethics committee approval .

• Outcome Measurements: Include comprehensive outcome measurements such as muscle fiber typing, satellite cell quantification, and muscle function assessments. Studies have shown that expression of myogenic factors impacts the density of Paired Box 7-positive cells and Myogenic factor 5-positive cells at different timepoints .

These considerations help ensure robust, reproducible results when working with recombinant myogenin in vivo.

How should researchers interpret contradictory results when studying Myogenin function across different species?

When confronted with contradictory results when studying Myogenin function across different species, researchers should employ the following analytical framework:

• Sequence Homology Analysis: Begin by comparing the sequence homology between species. While bovine and human myogenic factors share 96% homology in amino acid sequences, even small differences may affect function . Create detailed sequence alignments to identify specific differences that might explain functional disparities.

• Expression Pattern Comparison: Analyze whether expression patterns differ between species. For instance, while myogenin expression is muscle-specific across species, the timing and regulation may vary, affecting experimental outcomes.

• Regulatory Network Mapping: Map species-specific differences in regulatory networks. The interaction between myogenin and other factors (like MyoD, Ddx17, and PCAF) may vary across species .

• Methodological Standardization: Standardize experimental methodologies across species comparisons. Differences in cell culture conditions, reagent quality, or analytical techniques can lead to apparent contradictions.

• Contextual Interpretation: Interpret results within the proper developmental and physiological context. For example, muscle fiber type composition varies significantly across species, which may impact how myogenin influences fiber type transitions .

By systematically analyzing these factors, researchers can better understand whether contradictory results represent true biological differences or methodological variations.

What are promising areas for future research on recombinant Bovine Myogenin?

Several promising research directions for recombinant Bovine Myogenin warrant further investigation:

• Therapeutic Applications: Explore the potential therapeutic applications of myogenin in treating muscle wasting conditions. Research has shown that modulation of myogenin-associated pathways (such as Myoparr) can rescue muscle wasting in denervation models .

• Agricultural Applications: Investigate how manipulation of myogenin expression might improve livestock muscle development and meat quality. Studies with rbST have already demonstrated effects on myosin heavy chain composition and beta-adrenergic receptor expression in feedlot heifers .

• Single-Cell Dynamics: Utilize advanced imaging techniques and reporter systems (like the MyogntdTom mouse) to study the dynamics of myogenin expression at the single-cell level during muscle differentiation and regeneration .

• Long Non-coding RNA Interactions: Further characterize the interaction between myogenin and associated lncRNAs like Myoparr, exploring their potential as regulatory targets .

• CRISPR-Based Therapeutics: Develop CRISPR-based approaches to modulate myogenin expression or function in vivo, potentially offering new treatments for muscular disorders.

These research directions not only advance our fundamental understanding of myogenin biology but also offer potential applications in medicine and agriculture.

What technological advances might enhance research on Myogenin function?

Emerging technologies that could significantly advance Myogenin research include:

• Advanced Genome Editing: Next-generation CRISPR systems with improved specificity and efficiency will enable more precise manipulation of myogenin and its regulatory elements. Base editing and prime editing technologies offer the potential for single-nucleotide modifications without double-strand breaks .

• Intravital Imaging: Enhanced intravital microscopy techniques combined with reporter systems (like MyogntdTom) allow real-time visualization of myogenin expression dynamics in living tissues, providing insights into temporal regulation that static analyses cannot capture .

• Single-Cell Multi-omics: Integration of single-cell transcriptomics, proteomics, and epigenomics will provide comprehensive views of how myogenin functions within heterogeneous muscle cell populations during development and regeneration.

• Protein Interaction Mapping: Advanced protein-protein interaction mapping technologies, including proximity labeling methods, will help elucidate the complete myogenin interactome in different cellular contexts.

• Computational Modeling: Development of computational models integrating multiple data types will help predict myogenin function in complex physiological and pathological scenarios, guiding experimental design and interpretation.

These technological advances promise to overcome current limitations in myogenin research, enabling more comprehensive understanding of its regulatory roles and potential applications.