Recombinant Mouse Uncharacterized protein C8orf34 homolog

What is the mouse C8orf34 homolog and where is it localized within cells?

The mouse C8orf34 homolog (C1H8orf34) is a protein encoded by the C1H8orf34 gene located on chromosome 1 in mice, in contrast to its human counterpart located on chromosome 8 . Based on homology to the human protein, it is likely localized to the nucleus and nucleoli where it may play roles in gene expression regulation and cell cycle progression . The protein remains classified as "uncharacterized," indicating its full functions have yet to be elucidated. Multiple isoforms have been identified through alternative splicing, suggesting diverse functional roles in different cellular contexts .

How conserved is the C8orf34 protein across species compared to the mouse homolog?

The mouse C8orf34 homolog demonstrates high evolutionary conservation, sharing approximately 90.71% sequence identity with the human version . This substantial conservation suggests important biological functions maintained throughout mammalian evolution. The ortholog data shows the protein is well-preserved among mammals:

This conservation pattern indicates the protein likely emerged first in early metazoans, with significant functional refinement occurring in the vertebrate lineage .

What are the isoforms of mouse C8orf34 homolog identified to date?

Multiple isoforms of the mouse C8orf34 homolog have been documented in Mus caroli (Ryukyu mouse). According to GenScript database information, at least 11 different isoforms (X1-X11) have been identified . These isoforms result from alternative splicing events, suggesting complex transcriptional regulation. The complete list includes:

The diversity of isoforms suggests potential tissue-specific expression patterns and functional specialization .

What expression systems are most effective for producing recombinant mouse C8orf34 homolog?

For recombinant production of mouse C8orf34 homolog, researchers should consider multiple expression systems based on experimental requirements:

For optimal results, incorporate the following strategies:

• Use affinity tags (His, FLAG, GST) to facilitate purification

• Consider solubility-enhancing fusion partners (SUMO, MBP) to improve yield

• Optimize codon usage for the expression host

• For difficult constructs, express individual domains rather than full-length protein

What are the recommended approaches for detecting endogenous mouse C8orf34 homolog expression?

Detection of endogenous mouse C8orf34 homolog requires complementary approaches at both RNA and protein levels:

RNA detection methods:

• RT-qPCR with isoform-specific primers to quantify transcript levels across tissues

• RNA-seq for genome-wide expression profiling and detection of all splice variants

• RNA in situ hybridization for spatial localization in tissue sections

Protein detection methods:

• Western blotting with validated antibodies specific to conserved epitopes

• Immunohistochemistry or immunofluorescence for cellular/subcellular localization

• Proximity ligation assays to detect protein-protein interactions in situ

When analyzing multiple tissue types, particular attention should be paid to tissues where human C8orf34 shows clinical relevance, such as kidney (associated with papillary renal carcinoma) and auditory tissues (associated with hearing impairment) .

How can researchers validate knockout or knockdown models for mouse C8orf34 homolog?

Robust validation of C8orf34 homolog knockout or knockdown models requires a multi-level verification approach:

• Genomic verification: Confirm genetic modification using PCR and sequencing to verify precise deletion or mutation events.

• Transcript verification: Employ RT-qPCR targeting multiple regions of the transcript to ensure complete elimination of all functional isoforms. RNA-seq can provide comprehensive confirmation and reveal any compensatory changes in related genes.

• Protein verification: Perform Western blotting with validated antibodies to confirm absence of protein. Consider using multiple antibodies targeting different epitopes to ensure detection of all potential truncated products.

• Functional verification: Demonstrate phenotypic changes consistent with predicted function. For C8orf34, which may be involved in cell cycle regulation and gene expression, analyze cell proliferation, cell cycle progression, and transcriptional profiles in knockout/knockdown cells .

• Rescue experiments: Reintroduce wildtype or mutant versions of C8orf34 to demonstrate specific effects of the knockout and rule out off-target effects.

Potential pitfalls include incomplete knockout of all isoforms, compensatory mechanisms, and developmental adaptations that may mask phenotypes .

What evidence exists for C8orf34 homolog's role in cell cycle regulation?

The potential role of C8orf34 homolog in cell cycle regulation is suggested by several lines of evidence:

• The human ortholog interacts with MCM7, which is crucial for DNA replication initiation during the S phase of the cell cycle .

• C8orf34's interaction with ubiquitin C suggests involvement in protein degradation pathways that are essential for cell cycle progression, particularly the regulated degradation of cyclins .

• The protein's localization to the nucleus and nucleoli places it in cellular compartments integral to cell cycle control .

To investigate the specific role of mouse C8orf34 homolog in cell cycle regulation, researchers should consider:

• Synchronizing cells at different cell cycle phases and measuring C8orf34 expression and localization

• Analyzing cell cycle progression in C8orf34 knockout or overexpression models

• Performing chromatin immunoprecipitation to identify potential cell cycle-regulated genes under C8orf34 control

• Investigating phosphorylation or other post-translational modifications of C8orf34 during different cell cycle phases

Given its interaction with MCM7, particular attention should be paid to S-phase events and replication licensing mechanisms .

How does C8orf34 homolog contribute to gene expression regulation?

C8orf34 homolog likely contributes to gene expression regulation through several potential mechanisms:

• Nucleolar functions: The localization to nucleoli suggests potential involvement in ribosome biogenesis or other nucleolar activities that impact global protein synthesis and cell growth .

• Transcriptional regulation: Nuclear localization suggests possible roles in direct or indirect regulation of gene transcription, potentially through interaction with transcription factors or chromatin modifiers.

• Protein stability control: Interaction with ubiquitin C indicates potential involvement in regulating the stability of transcription factors or other gene expression regulators through the ubiquitin-proteasome system .

To investigate these mechanisms, researchers can:

• Perform RNA-seq after C8orf34 knockdown/overexpression to identify affected gene networks

• Use ChIP-seq to identify genomic binding sites

• Conduct proteomics analyses to identify changes in protein abundance after C8orf34 manipulation

• Employ reporter gene assays to test direct effects on transcription of specific genes

These approaches can help elucidate whether C8orf34 functions as a direct transcriptional regulator or indirectly affects gene expression through other mechanisms.

What is known about C8orf34 homolog's role in disease models and clinical applications?

C8orf34 has been implicated in several human disease contexts that suggest important research directions for mouse models:

• Cancer associations: A translocation causing fusion of human C8orf34 with the MET protooncogene has been identified in papillary renal carcinoma . Additionally, C8orf34-as1 (antisense RNA 1) has been implicated in a ceRNA regulatory axis in lung adenocarcinoma, where it interacts with miR-671-5p and MFAP4 .

• Treatment response: Variants in C8orf34 (rs1517114) have been associated with risk for diarrhea and neutropenia in patients receiving chemotherapy . This genetic marker has demonstrated 100% concordance in analytical validation studies for genotype-guided therapy .

• Developmental disorders: Mutations in C8orf34 have been linked to congenital hearing impairment .

For mouse models investigating these conditions, researchers should:

• Generate transgenic models expressing C8orf34-MET fusion proteins to study mechanisms of renal carcinogenesis

• Develop mouse models with the equivalent of human rs1517114 to study chemotherapy toxicity

• Create conditional knockout models targeting auditory tissues to investigate hearing development

These disease associations highlight the potential clinical significance of understanding C8orf34 homolog function and regulation.

How can researchers design experiments to investigate C8orf34 homolog in cancer models?

Based on the identified connections between C8orf34 and cancer, researchers can design experimental approaches to investigate its role in oncogenesis:

• Expression analysis in cancer models:

  • Analyze C8orf34 expression across different mouse cancer models, focusing on renal and lung cancers

  • Compare expression in tumor vs. normal tissues and correlate with tumor progression markers

  • Investigate isoform-specific expression patterns in different cancer types

• Functional studies:

  • Generate C8orf34 knockout or overexpression in cancer cell lines to assess effects on proliferation, migration, and invasion

  • Develop mouse models with conditional C8orf34 manipulation in specific tissues (kidney, lung) to observe tumor development

  • Engineer models expressing the C8orf34-MET fusion protein identified in human renal carcinomas

• Mechanistic investigations:

  • Analyze C8orf34's interaction with known tumor suppressors, particularly MTUS2

  • Investigate the C8orf34-as1/miR-671-5p/MFAP4 regulatory axis identified in lung adenocarcinoma

  • Examine effects on cell cycle regulation through interaction with MCM7 and ubiquitin pathway components

• Therapeutic targeting:

  • Screen for molecules that modulate C8orf34 activity or expression

  • Test combination approaches targeting C8orf34 and its interaction partners

  • Evaluate C8orf34 as a biomarker for treatment response

These approaches can help establish whether C8orf34 functions as an oncogene, tumor suppressor, or modifier gene in different cancer contexts.

What genetic variants in mouse C8orf34 homolog might correspond to clinically relevant human variants?

The identification of human C8orf34 genetic variants with clinical significance provides direction for corresponding mouse studies:

• rs1517114: This variant has been associated with chemotherapy-induced toxicity in humans . Researchers can:

  • Identify the equivalent nucleotide position in mouse C8orf34 homolog

  • Generate knock-in models with the corresponding mutation using CRISPR-Cas9

  • Evaluate these models for altered sensitivity to chemotherapeutic agents

• C8orf34-MET fusion: The translocation identified in human papillary renal carcinoma can be modeled by:

  • Creating transgenic mice expressing the fusion protein under kidney-specific promoters

  • Developing cell line models expressing the fusion protein to study altered signaling pathways

  • Using CRISPR-based approaches to engineer the chromosomal translocation in mouse cells

• Mutations associated with hearing impairment: Researchers should:

  • Screen for conserved domains between human and mouse proteins

  • Target these regions for mutation in mouse models

  • Conduct comprehensive auditory phenotyping of resulting models

Analytical validation of genetic testing for C8orf34 variants has shown 100% concordance in human studies , suggesting robust methods can be developed for detecting equivalent mouse variants.

What are the major challenges in purifying recombinant mouse C8orf34 homolog and how can they be addressed?

Purification of recombinant mouse C8orf34 homolog presents several technical challenges typical of nuclear proteins:

• Solubility issues: Nuclear proteins often form inclusion bodies during recombinant expression. Solutions include:

  • Using solubility-enhancing fusion tags (SUMO, MBP, GST)

  • Optimizing expression conditions (lower temperature, reduced inducer concentration)

  • Employing on-column refolding techniques

  • Testing different detergents or solubilizing agents

• Protein stability: To enhance stability during purification:

  • Optimize buffer conditions (pH, salt concentration, reducing agents)

  • Include protease inhibitors throughout purification

  • Test addition of stabilizing agents (glycerol, arginine, trehalose)

  • Perform thermal shift assays to identify stabilizing conditions

• Nucleic acid contamination: Nuclear proteins often bind DNA/RNA non-specifically:

  • Include nuclease treatment steps

  • Use high salt washes during affinity purification

  • Consider ion exchange chromatography to separate protein-nucleic acid complexes

• Post-translational modifications: If functional studies require native modifications:

  • Express in mammalian or insect cells rather than bacterial systems

  • Consider phosphatase inhibitors if phosphorylation is suspected

  • Analyze purified protein by mass spectrometry to verify modification status

The availability of cDNA ORF clones for mouse C8orf34 homolog provides a starting point for designing expression constructs optimized for specific purification strategies .

How can researchers effectively study the interactions between C8orf34 homolog and its binding partners?

To comprehensively characterize C8orf34 homolog interactions with binding partners, researchers should employ complementary approaches:

• In vitro binding assays:

  • Surface Plasmon Resonance (SPR) to determine binding kinetics and affinity

  • Isothermal Titration Calorimetry (ITC) to measure thermodynamic parameters

  • Pull-down assays with purified components to confirm direct interactions

  • Protein microarray screening to identify novel binding partners

• Cellular interaction studies:

  • Bimolecular Fluorescence Complementation (BiFC) for visualizing interactions in live cells

  • Förster Resonance Energy Transfer (FRET) to detect close proximity in real-time

  • Proximity Ligation Assay (PLA) to visualize endogenous protein interactions in situ

  • Co-immunoprecipitation from different cellular compartments and conditions

• Structural characterization:

  • X-ray crystallography of co-crystals with binding partners

  • Cryo-EM for larger complexes

  • NMR spectroscopy to map interaction interfaces

  • Hydrogen-deuterium exchange mass spectrometry to identify binding regions

• Network analysis:

  • Integrate protein interaction data with transcriptomic responses

  • Map interactions onto known signaling pathways

  • Identify hub proteins that may coordinate C8orf34 functions

Based on human C8orf34's interactions with ubiquitin C, MTUS2, and MCM7 , these partners should be prioritized for initial interaction studies with the mouse homolog.

What advanced genomic approaches can reveal C8orf34 homolog's regulatory networks?

To elucidate the regulatory networks involving C8orf34 homolog, researchers can apply several cutting-edge genomic approaches:

• ChIP-sequencing and variants:

  • Standard ChIP-seq to identify DNA binding sites

  • CUT&RUN or CUT&Tag for higher resolution with less material

  • ChIP-exo for base-pair resolution of binding sites

  • ChIP-seq with synchronized cells to capture cell cycle-specific interactions

• Transcriptome analysis:

  • RNA-seq comparing wildtype and C8orf34 knockout models

  • Single-cell RNA-seq to identify cell-type specific effects

  • Nascent RNA sequencing (GRO-seq, PRO-seq) to distinguish direct transcriptional effects

  • Alternative splicing analysis to identify regulatory roles in RNA processing

• Chromatin structure analysis:

  • ATAC-seq to identify regions of open chromatin

  • Hi-C or ChIA-PET to map three-dimensional chromatin interactions

  • CUT&RUN with histone modification antibodies to assess epigenetic changes

• Integrative approaches:

  • Multi-omics integration of proteomics and transcriptomics data

  • Network analysis to identify key nodes in regulatory networks

  • Comparative analysis across different tissues and developmental stages

These approaches can help determine whether C8orf34 homolog functions primarily in transcriptional regulation, chromatin organization, or post-transcriptional processes, providing a comprehensive view of its regulatory impact.