Recombinant Mouse UPF0704 protein C6orf165 homolog

What is the UPF0704 protein C6orf165 homolog and what are its known functions?

The UPF0704 protein C6orf165 homolog is part of a family of proteins found in various organisms. As homologs (genes related to each other by descent from a common ancestor), these proteins may have similar functions across different species, though their exact roles can vary. Research indicates that homologous proteins like CTF4 are specifically expressed in actively dividing cells and young tissues, suggesting potential roles in cell proliferation and development. In yeast, the related CTF4 protein suppresses the formation of DNA double-strand breaks, indicating a potential role in DNA maintenance and genome integrity.

What database identifiers are associated with mouse UPF0704 protein C6orf165 homolog?

The mouse UPF0704 protein C6orf165 homolog can be located in major biological databases using the following identifiers:

These identifiers allow researchers to access comprehensive information about the protein's sequence, structure, and potential interactions for further investigation.

How is UPF0704 protein related to other homologous proteins in different organisms?

UPF0704 protein C6orf165 homolog is evolutionarily related to proteins in other organisms that may serve similar biological functions. In plants, homologous CTF4 proteins interact with Polycomb Repressive Complex 2 (PRC2) components CLF and SWN, as well as the PRC1 component LHP1, suggesting roles in epigenetic regulation. The rice dwarf-related wd40 protein 1 (DRW1), another homolog, physically interacts with subunits of a DNA helicase complex and DNA polymerase α, which are essential for DNA replication and prevention of DNA damage. These relationships indicate that UPF0704 protein C6orf165 homolog may participate in conserved biological processes related to DNA maintenance, replication, and potentially cell division.

What are the recommended protocols for expressing and purifying recombinant mouse UPF0704 protein?

While the search results don't provide specific purification protocols for UPF0704 protein C6orf165 homolog, researchers can adapt standard recombinant protein methodologies similar to those used for related proteins such as Dkk-1 :

• Expression System Selection: Choose between bacterial (E. coli), mammalian, or insect cell expression systems based on requirements for post-translational modifications.

• Vector Design: Incorporate affinity tags (His, GST, or FLAG) to facilitate purification while ensuring they don't interfere with protein function.

• Purification Strategy:

  • Initial capture using affinity chromatography

  • Secondary purification via ion exchange or size exclusion chromatography

  • Quality control through SDS-PAGE and Western blotting

• Reconstitution: Prepare the purified protein in an appropriate buffer, considering stability requirements and downstream applications.

The choice of expression system should consider that mammalian proteins often require proper folding and post-translational modifications for biological activity, which might necessitate mammalian or insect cell expression systems rather than bacterial systems.

How can researchers generate knockout models to study UPF0704 protein function?

Based on successful approaches used for studying related proteins, researchers can implement the following strategy to generate knockout models for UPF0704 protein C6orf165 homolog :

• CRISPR-Cas9 Design:

  • Design guide RNAs targeting exons of the UPF0704 gene

  • Validate guide RNA specificity using bioinformatic tools to minimize off-target effects

  • Consider generating frameshift mutations for complete protein knockout

• Model Organism Selection:

  • Mouse models are appropriate given the established relevance of related CFAP206 knockout studies in mice

  • Cell line models can provide preliminary functional data

• Verification of Knockout Efficiency:

  • Confirm gene editing through DNA sequencing

  • Verify protein absence using Western blotting or immunostaining

  • Assess mRNA levels using RT-qPCR

• Phenotypic Analysis:

  • Cell proliferation and division dynamics

  • DNA damage response assays

  • Tissue-specific expression patterns and associated phenotypes

This approach follows established methodologies that proved successful in characterizing related proteins like CFAP206 .

What immunostaining approaches are effective for detecting UPF0704 protein in tissue samples?

Based on successful strategies employed for related proteins, researchers should consider the following immunostaining approach for UPF0704 protein C6orf165 homolog detection:

• Sample Preparation:

  • Fixation: 4% paraformaldehyde for 15-20 minutes for cells; 24 hours for tissue sections

  • Permeabilization: 0.2% Triton X-100 in PBS for 10 minutes

  • Antigen retrieval: Citrate buffer pH 6.0 heating for tissue sections

• Staining Protocol:

  • Blocking: 5% normal serum (species-dependent on secondary antibody) for 1 hour

  • Primary antibody: Anti-UPF0704 protein (1:100-1:500 dilution, optimization required)

  • Secondary antibody: Species-appropriate fluorophore-conjugated antibody

  • Nuclear counterstain: DAPI (1:1000)

• Co-localization Studies:

  • Consider co-staining with markers for actively dividing cells

  • Include markers for DNA replication machinery given potential roles in DNA maintenance

• Controls:

  • Positive control: Tissues known to express UPF0704 (based on actively dividing cells)

  • Negative control: UPF0704 knockout tissues or antibody omission

  • Specificity control: Pre-absorption with recombinant protein

This approach parallels methods shown to be effective for CFAP206 detection in sperm cells, where immunostaining successfully demonstrated protein localization and functional relationships .

How can protein-protein interaction studies be designed to identify binding partners of UPF0704 protein?

To comprehensively identify binding partners of UPF0704 protein C6orf165 homolog, researchers should implement a multi-technique approach:

• Affinity Purification-Mass Spectrometry (AP-MS):

  • Express tagged UPF0704 protein in appropriate cell lines

  • Perform pull-down using tag-specific antibodies

  • Identify binding partners through mass spectrometry

  • Validate key interactions with co-immunoprecipitation and Western blotting

• Proximity-Based Labeling:

  • Generate BioID or TurboID fusion constructs with UPF0704

  • Express in cell models to enable biotinylation of proximal proteins

  • Capture biotinylated proteins with streptavidin and identify by MS

  • Distinguish between direct interactions and proximity-based associations

• Yeast Two-Hybrid Screening:

  • Use UPF0704 as bait against cDNA libraries

  • Focus on tissue-specific libraries from actively dividing cells

  • Validate positive interactions in mammalian cell systems

Based on homologous protein interactions, key proteins to evaluate include:

• DNA helicase complex components

• DNA polymerase α and associated factors

• PRC1 and PRC2 complex components (in appropriate contexts)

This approach is informed by successful interaction studies of related proteins where CTF4 homologs in plants were shown to interact with specific chromatin regulatory components.

What are the recommended approaches for studying the role of UPF0704 protein in DNA damage response?

Given the potential involvement of UPF0704 protein C6orf165 homolog in DNA maintenance (based on homologous proteins), researchers should consider these approaches:

• DNA Damage Induction and Response Assessment:

  Damage Type	Induction Method	Response Measurement
  Double-strand breaks	Ionizing radiation (2-10 Gy)	γ-H2AX foci formation
  Replication stress	Hydroxyurea (0.5-2 mM)	RPA foci, phospho-CHK1
  Crosslinking damage	Mitomycin C (10-100 ng/ml)	FANCD2 ubiquitination

• Comparative Analysis in Wild-Type vs. UPF0704-Deficient Models:

  • Measure repair kinetics through time-course experiments

  • Assess checkpoint activation through phospho-specific antibodies

  • Quantify chromosomal abnormalities after damage

• Replication Fork Stability Analysis:

  • DNA fiber assay to measure fork progression and restart

  • iPOND (isolation of Proteins On Nascent DNA) to identify UPF0704 at replication forks

  • Assessment of fork collapse in response to replication stress

• Epistasis Analysis:

  • Generate double knockouts with known DNA repair factors

  • Assess synthetic lethality or rescue phenotypes

  • Place UPF0704 in established DNA damage response pathways

This experimental strategy leverages the observation that CTF4 in yeast suppresses the formation of DNA double-strand breaks, suggesting similar potential functions for UPF0704 protein C6orf165 homolog in mammals.

How can researchers investigate the potential role of UPF0704 protein in ciliary development, given its homology to CFAP206?

Based on the relationship between UPF0704 protein C6orf165 homolog and CFAP206 (a protein associated with ciliary development) , researchers can investigate potential roles in ciliary formation through these approaches:

• Expression Analysis in Ciliated Tissues:

  • RT-qPCR quantification in tissues with motile cilia (respiratory epithelium, ependymal cells)

  • Western blot analysis of protein expression during ciliogenesis

  • Temporal expression analysis during differentiation of primary ciliated cells

• Subcellular Localization Studies:

  • Immunofluorescence co-staining with ciliary markers (acetylated tubulin, Arl13b)

  • Super-resolution microscopy to define precise localization within ciliary structures

  • Electron microscopy to examine ultrastructural defects in knockout models

• Functional Assessment of Ciliary Motility:

  • High-speed video microscopy to analyze ciliary beat frequency and pattern

  • Mucociliary clearance assays in airway epithelial cultures

  • Particle tracking to quantify fluid flow generated by cilia

• Molecular Interactions with Ciliary Apparatus:

  • Proximity labeling to identify interactions with established ciliary proteins

  • Co-immunoprecipitation with radial spoke proteins and dynein arm components

The experimental design should be informed by the established methodology used to demonstrate that CFAP206 deficiency leads to abnormal ciliary beating and sperm flagellum defects , while adapting these approaches to investigate broader ciliary functions of UPF0704 protein.

How should researchers address data inconsistencies in UPF0704 protein localization studies?

When encountering conflicting data regarding UPF0704 protein localization, researchers should implement this systematic approach:

• Technical Validation:

  • Evaluate antibody specificity using knockout controls and Western blotting

  • Compare multiple fixation and permeabilization protocols that may affect epitope accessibility

  • Utilize multiple detection methods (immunofluorescence, electron microscopy, subcellular fractionation)

• Biological Context Analysis:

  • Assess localization across different cell types and developmental stages

  • Evaluate effects of cell cycle phase on protein localization

  • Consider stimulus-dependent translocation possibilities

• Resolution of Inconsistencies:

  • Implement quantitative image analysis with statistical validation

  • Consider dual-function possibilities with context-dependent localization

  • Evaluate post-translational modifications that might affect localization

• Integration Framework:

  Data Source	Strength of Evidence	Potential Confounding Factors	Integration Approach
  Immunofluorescence	Direct visualization	Antibody cross-reactivity	Multiple antibodies targeting different epitopes
  Fractionation/Western	Biochemical validation	Contamination between fractions	Multiple fractionation methods
  Tagged protein expression	Controlled system	Overexpression artifacts	Knock-in tags at endogenous loci
  Proximity labeling	In vivo context	Background biotinylation	Appropriate controls and statistical thresholds

By systematically addressing discrepancies through this framework, researchers can develop a more nuanced understanding of UPF0704 protein localization patterns across different biological contexts.

What are the considerations for computational prediction of UPF0704 protein function?

When employing computational approaches to predict UPF0704 protein C6orf165 homolog function, researchers should consider:

• Sequence-Based Prediction Methods:

  • Multiple sequence alignment with CTF4 homologs across species

  • Domain identification and functional prediction

  • Conservation analysis of specific residues/motifs

  • Evaluation of confidence scores for predictions

• Structural Prediction Approaches:

  • Template-based modeling using related proteins with known structures

  • Ab initio modeling for novel domains

  • Molecular dynamics simulations to predict functional movements

  • Binding site prediction for potential interactors

• Integration with Experimental Data:

  • Correlation of predictions with knockout phenotype data

  • Refinement based on protein-protein interaction data

  • Validation through directed mutagenesis of predicted functional sites

• Machine Learning Enhancement:

  • Implementation of approaches used in the GeneDisco framework for target validation

  • Active learning algorithms to prioritize experimental validations

  • Integration of diverse data types (expression, interaction, evolutionary)

• Functional Annotation Confidence Assessment:

  Prediction Type	Tools/Databases	Confidence Metrics	Validation Approach
  GO terms	InterPro, BLAST2GO	Statistical significance	Experimental validation of top predictions
  Protein-protein interactions	STRING, PrePPI	Confidence scores, conservation	Co-IP validation of high-confidence predictions
  Pathway involvement	KEGG, Reactome	Enrichment analysis	Targeted disruption of predicted pathway nodes
  Disease associations	DisGeNET, OMIM	Statistical association	Phenotypic analysis in disease models

This systematic approach builds upon established methodologies while recognizing the limitations of computational predictions, especially for proteins with limited experimental characterization like UPF0704.

How should researchers design experiments to differentiate between direct and indirect effects in UPF0704 knockout phenotypes?

To distinguish direct effects of UPF0704 protein deficiency from indirect consequences, researchers should implement this comprehensive strategy:

• Temporal Analysis of Phenotype Development:

  • Time-course studies following knockout/knockdown

  • Identification of primary versus secondary effects

  • Correlation with protein degradation kinetics in inducible systems

• Rescue Experiments with Precise Design:

  • Wild-type protein reintroduction

  • Domain-specific mutant complementation

  • Orthologous protein rescue from different species

  • Temporal control of rescue using inducible systems

• Molecular Target Validation:

  • ChIP-seq or CUT&RUN for chromatin-associated functions

  • RNA-seq time course to identify earliest transcriptional changes

  • Phosphoproteomics to identify signaling pathway alterations

  • Targeted analysis of pathways implicated in CTF4/CFAP functions

• Single-Cell Approaches for Heterogeneity Assessment:

  • Single-cell RNA-seq to identify cell populations most affected

  • Trajectory analysis to map progression of phenotypic changes

  • Spatial transcriptomics to identify tissue-specific effects

• Mechanistic Dissection Framework:

  Approach	Application	Outcome Interpretation
  Conditional knockout	Tissue/temporal specific deletion	Identifies direct tissue-specific requirements
  Separation-of-function mutations	Targeted disruption of specific interactions	Links specific interactions to phenotypes
  Acute vs. chronic depletion	Comparison of RNAi vs. genetic knockout	Distinguishes adaptive responses from direct effects
  Epistasis analysis	Double knockouts with pathway components	Places protein in functional pathways
  Cross-species complementation	Rescue with orthologs of varying divergence	Identifies evolutionarily conserved functions

This approach draws from successful strategies used in characterizing CFAP206 function in sperm flagellum development , where researchers distinguished direct structural roles from secondary consequences through careful experimental design and phenotypic analysis.

What is the potential relationship between UPF0704 protein function and human disease conditions?

While direct disease associations for UPF0704 protein C6orf165 homolog are not yet established, researchers can explore potential disease relevance based on:

• Analysis of Related Protein Disease Associations:

  • CFAP206 mutations are associated with male infertility due to sperm flagellum defects

  • CFAP206 has associations with Aleutian Mink Disease and Primary Ciliary Dyskinesia

  • Research on knockout mouse strains has identified numerous unannotated genes with strong metabolic phenotypes

• Potential Disease Categories for Investigation:

  Disease Category	Rationale	Research Approach
  Ciliopathies	Homology to ciliary protein CFAP206	Genotyping in ciliopathy cohorts without known mutations
  Male infertility	CFAP206 association with sperm flagellum	Analysis in infertility cases with flagellar defects
  DNA repair disorders	Homology to DNA maintenance proteins	Screening in patients with unexplained genome instability
  Metabolic disorders	Association with metabolic phenotypes	Analysis in cohorts with unexplained metabolic disease

• Functional Validation in Disease Models:

  • Analysis of patient-derived cells for UPF0704 expression/function

  • CRISPR-engineering of patient-specific mutations in cellular models

  • Phenotypic rescue experiments in patient-derived cells

This approach builds on the established relationship between CFAP206 deficiency and male infertility , while expanding investigation to other potential disease associations based on the functional relatedness to DNA maintenance and ciliary proteins.

How can researchers investigate the therapeutic potential of modulating UPF0704 protein activity?

To explore potential therapeutic applications related to UPF0704 protein modulation, researchers should consider:

• Target Validation Strategy:

  • Comprehensive phenotypic analysis of tissue-specific knockouts

  • Evaluation of haploinsufficiency vs. complete loss effects

  • Identification of specific pathways affected by UPF0704 deficiency

  • Assessment of temporal requirements through inducible systems

• Therapeutic Modulation Approaches:

  • Small molecule screening for protein stabilization/destabilization

  • Identification of critical protein-protein interactions for targeted disruption

  • Antisense oligonucleotide approaches for transcript modulation

  • Protein replacement strategies for loss-of-function scenarios

• Model Systems for Therapeutic Evaluation:

  • Patient-derived cells with relevant disease phenotypes

  • Organoid models to assess tissue-specific effects

  • CRISPR-engineered mouse models with human-equivalent mutations

  • Functional assays relevant to identified disease associations

• Translational Research Implementation:

  • Utilize active learning approaches from GeneDisco framework to prioritize experiments

  • Integrate prior knowledge from various information sources to guide therapeutic hypothesis generation

  • Develop assays suitable for high-throughput screening of potential modulators

This systematic approach parallels successful therapeutic development strategies for other proteins involved in DNA maintenance and ciliary function, while acknowledging the early stage of UPF0704 protein characterization.