Recombinant Mouse Uncharacterized protein C7orf26 homolog

What is known about the function of mouse C7orf26 homolog in relation to its human counterpart?

The mouse C7orf26 homolog shares significant functional similarities with the human C7orf26 gene, which has been identified in connection with autosomal dominant ocular disease and functions in mRNA splicing . Based on gene set enrichment analysis of human C7orf26, the protein is expected to function with the integrator complex in mRNA splicing mechanisms. When C7orf26 expression was knocked down in human cells, approximately 700 transcripts were downregulated by 50% or more, with genes involved in "alternative splicing" or "splice variant" functions representing a significant proportion of these affected transcripts . The mouse homolog likely plays similar roles in splicing regulation, though species-specific differences may exist in target gene profiles.

What expression systems are most effective for producing recombinant mouse C7orf26 homolog?

Based on successful expression systems for other recombinant mouse proteins, HEK293 cells represent an efficient mammalian expression system for mouse C7orf26 homolog . This system allows for proper post-translational modifications and folding of mammalian proteins. For optimal expression:

• Clone the full-length mouse C7orf26 homolog cDNA into a mammalian expression vector containing a strong promoter (CMV/EF1α)

• Consider adding a purification tag (His, FLAG, or Fc fusion) for downstream isolation

• Transfect HEK293 cells using calcium phosphate precipitation or lipid-based transfection reagents

• Select stable transfectants if long-term production is desired

• Harvest and purify using affinity chromatography appropriate for the fusion tag

Alternative systems like bacterial expression (E. coli) may be suitable for producing truncated domains but typically yield lower functional protein quality for full-length mammalian proteins .

How can I validate the expression and purity of recombinant mouse C7orf26 homolog?

A comprehensive validation protocol should include:

For proteins like C7orf26 involved in splicing, functional validation through in vitro splicing assays or reporter-based cellular assays is particularly important to ensure biological activity is preserved in the recombinant form .

What are the methodological considerations for homologous recombination targeting mouse C7orf26?

Homologous recombination targeting of mouse C7orf26 requires careful consideration of several factors:

• Vector Design: Create a targeting vector with 5-10kb homology arms flanking the C7orf26 locus. Include a positive selection marker (e.g., neomycin resistance) and potentially a negative selection marker (e.g., thymidine kinase) for enrichment of correctly targeted cells .

• Electroporation Protocol: Standard mouse ES cell electroporation protocols may require optimization. For example, human ES cells required modified electroporation approaches based on their physical characteristics . Similarly, mouse ES cells for C7orf26 targeting may need calibrated parameters:

  • Cell density: 1-5 × 10^7 cells/ml

  • Voltage: 220-240V

  • Capacitance: 475-500μF

  • Time constant: 8-12ms

• Screening Strategy: Design PCR primers or Southern blot probes that can distinguish between random integration and targeted homologous recombination events .

• Verification: Confirm targeting through sequencing and functional assays to ensure the desired modification affects C7orf26 expression or function as intended.

How does mouse C7orf26 homolog potentially interact with the integrator complex in mRNA splicing?

Based on studies with human C7orf26, the mouse homolog is likely involved in the integrator complex-mediated RNA processing pathway . Potential interaction mechanisms include:

• Direct Binding: C7orf26 may directly associate with core components of the integrator complex (INT1-14)

• Regulatory Function: The protein may serve as a regulatory subunit that modulates integrator activity in response to cellular signals

• Substrate Recognition: C7orf26 could assist in recognition of specific pre-mRNA substrates, particularly those involved in ocular development given its association with ocular disease

To experimentally investigate these interactions:

• Conduct co-immunoprecipitation assays with tagged mouse C7orf26 followed by mass spectrometry

• Perform yeast two-hybrid or proximity labeling experiments to identify direct binding partners

• Use CLIP-seq (Cross-linking immunoprecipitation sequencing) to identify RNA targets

What assays can confirm the functional activity of recombinant mouse C7orf26 homolog?

Several complementary approaches can verify functional activity:

• In vitro splicing assays: Using synthetic pre-mRNA substrates and nuclear extracts supplemented with recombinant C7orf26 to assess impact on splicing efficiency and accuracy

• Minigene splicing reporters: Transfect cells with reporter constructs containing exons separated by an intron, along with recombinant C7orf26, and analyze splicing patterns

• Rescue experiments: Complement C7orf26 knockdown cells with recombinant protein and assess restoration of normal splicing patterns for the 700+ transcripts known to be affected by C7orf26 depletion

• Integrator complex assembly assays: Analyze whether recombinant C7orf26 can incorporate into the integrator complex using sucrose gradient fractionation or native gel electrophoresis

What mouse models are most suitable for studying C7orf26 homolog function?

Several mouse model approaches can be employed to study C7orf26 function:

• Conditional knockout models: Using Cre-loxP system to delete C7orf26 in specific tissues or developmental stages, particularly in ocular tissues given the connection to eye development

• Reporter knock-in models: Inserting fluorescent reporters like GFP while maintaining C7orf26 expression to track tissue distribution and subcellular localization

• Tolerant transgenic models: For studies involving transfer of C7orf26-modified cells, using a mouse strain like the 'Tol' model that prevents immune rejection of reporter-protein modified cells through physiological self-tolerance mechanisms

When generating these models, consider the immunological aspects of introducing modified proteins. The 'Tol' mouse model approach demonstrates that expression of a transgene can result in deletion of CD8+ T cells specific for model epitopes, substantially improving engraftment of gene-modified cells .

How can I investigate the role of mouse C7orf26 homolog in ocular development?

Given the connection between human C7orf26 and autosomal dominant ocular disease , investigating its mouse homolog in eye development requires:

• Temporal expression analysis:

  • RT-qPCR of C7orf26 across different developmental stages of mouse eye tissues

  • In situ hybridization to localize expression in specific ocular structures

• Conditional knockout approach:

  • Generate Cre-driver lines specific to ocular tissues (lens, retina, cornea)

  • Analyze phenotypes including histology, immunohistochemistry, and functional tests (ERG, OCT)

• Transcriptomic profiling:

  • Compare RNA-seq data from wild-type and C7orf26-deficient ocular tissues

  • Focus on alternative splicing events in eye development genes

  • Analyze enriched GO terms similar to those identified in the human C7orf26 knockdown study

• Rescue experiments:

  • Test whether human C7orf26 can compensate for mouse C7orf26 deficiency

  • Introduce structure-function mutants to identify critical domains

What bioinformatics approaches can help characterize mouse C7orf26 homolog function?

Comprehensive bioinformatic analysis should include:

• Comparative Genomics:

  • Analyze conservation patterns across species to identify functionally important domains

  • Compare human and mouse C7orf26 for conserved motifs potentially related to RNA binding or protein interaction

• Structural Prediction:

  • Use AlphaFold or RoseTTAFold to predict 3D structure

  • Identify potential functional domains based on structural homology

• Interaction Network Analysis:

  • Create protein-protein interaction networks centered on C7orf26 and integrator complex components

  • Perform pathway enrichment analysis to identify biological processes potentially affected by C7orf26

• RNA-Seq Data Analysis:

  • Process data similar to the human C7orf26 knockdown study that identified 700 downregulated transcripts

  • Use rMATS or similar tools to detect differential alternative splicing events

  • Analyze intron retention patterns and exon usage

• Motif Analysis:

  • Identify sequence motifs in pre-mRNAs that might be preferentially affected by C7orf26 activity

  • Compare these to known binding motifs of splicing regulators

How should I interpret differential gene expression following C7orf26 knockdown or knockout?

When analyzing differential gene expression data following C7orf26 manipulation:

• Primary vs. Secondary Effects:

  • Primary effects likely include genes directly affected by C7orf26's role in mRNA splicing

  • Secondary effects may reflect downstream consequences of misregulated splicing

• Temporal Analysis:

  • Perform time-course experiments to distinguish immediate from delayed effects

  • Early response genes (24-48h post-knockdown) more likely represent direct targets

• Functional Classification:

  • Apply GO term enrichment analysis similar to the human study that showed enrichment for "alternative splicing" or "splice variant" terms

  • Cluster affected genes by biological process to identify key pathways

• Splicing-Specific Analysis:

  • Beyond simple differential expression, analyze:

    • Exon inclusion/exclusion rates

    • Intron retention events

    • Alternative 5' or 3' splice site usage

    • Alternative promoter or polyadenylation site selection

• Integration with Other Data Types:

  • Correlate expression changes with C7orf26 binding sites from CLIP-seq

  • Cross-reference with integrator complex binding data

What are common challenges in producing active recombinant mouse C7orf26 homolog and how can they be addressed?

For C7orf26 specifically, consider:

• Purifying together with known binding partners from the integrator complex

• Including RNA during purification if RNA-binding is essential for activity

• Testing different tags and their positions (N-terminal vs. C-terminal)

How can I optimize transfection efficiency for studies involving mouse C7orf26 homolog?

Achieving high transfection efficiency is crucial for functional studies:

• Cell Type Optimization:

  • For mouse embryonic stem cells, electroporation protocols must be carefully calibrated as standard conditions work poorly in human ES cells

  • For fibroblasts or HEK293 cells, lipid-based transfection typically works well

• DNA Quality Considerations:

  • Use endotoxin-free plasmid preparations

  • Maintain optimal DNA concentration (typically 0.5-1 μg/μl)

  • Ensure appropriate vector size (smaller constructs typically transfect more efficiently)

• Transfection Parameter Optimization:

  • For electroporation: systematically test voltage, capacitance, and pulse duration

  • For chemical transfection: optimize DNA:reagent ratios, incubation times, and cell density

• Verification Methods:

  • Include reporter genes (GFP, luciferase) to monitor transfection efficiency

  • Use immunoblotting or qPCR to confirm C7orf26 expression levels

  • Consider dual-reporter systems to normalize for transfection efficiency variations

• Stable Cell Line Generation:

  • For long-term studies, develop stable cell lines expressing mouse C7orf26 homolog

  • Consider inducible expression systems (Tet-On/Off) if constitutive expression is toxic