Recombinant Mouse Uncharacterized protein KIAA0146 (Kiaa0146), partial

What is KIAA0146 and what is its function in DNA repair mechanisms?

KIAA0146, also known as SPIDR (Scaffolding protein involved in DNA repair), functions as a critical modulator in DNA damage response pathways. While its precise biochemical function remains to be fully characterized, SPIDR has been identified as a key interacting partner of several essential DNA repair proteins, including RAD51, SWSAP1-SWS1, and the BLM helicase . These interactions suggest that SPIDR plays a scaffolding role in organizing protein complexes involved in high-fidelity DNA repair mechanisms. Current research indicates that SPIDR contributes to genome stability, as cellular analyses of SPIDR mutations have demonstrated elevated levels of homologous recombination, single-strand annealing, and non-homologous end joining compared to wild-type cells . These findings highlight SPIDR's importance in maintaining genomic integrity through its involvement in DNA repair processes.

How does mouse KIAA0146 compare structurally to its human ortholog?

The mouse KIAA0146 (SPIDR) ortholog shares significant sequence homology with the human version, particularly in functional domains involved in protein-protein interactions. Researchers should note that while the core functional regions demonstrate high conservation between species, there may be subtle differences in regulatory elements and post-translational modification sites. When designing experiments using recombinant mouse KIAA0146 as a model for human studies, investigators should perform careful sequence alignments to identify conserved domains and potential species-specific variations. This comparative analysis becomes particularly important when interpreting results from mouse models in the context of human pathologies such as primary ovarian insufficiency (POI) . Understanding these structural similarities and differences is essential for accurately translating findings between mouse models and human disease mechanisms.

What experimental systems are most appropriate for studying KIAA0146 function?

For investigating KIAA0146/SPIDR function, researchers can employ multiple complementary experimental systems. Cell-based assays using mouse embryonic fibroblasts (MEFs) or mouse ovarian cell lines provide valuable insights into protein localization, interaction networks, and responses to DNA damage. For in vivo studies, both conventional knockout mice and conditional SPIDR knockout models can reveal tissue-specific functions, particularly in reproductive tissues where SPIDR mutations have been implicated in fertility disorders . When studying specific mutations identified in human patients (such as premature stop codons), CRISPR/Cas9-mediated genome editing can generate precise mouse models harboring equivalent mutations. Additionally, biochemical approaches using purified recombinant mouse KIAA0146 protein enable detailed analysis of direct protein interactions, enzymatic activities, and structural studies. A multi-system approach combining these methods provides the most comprehensive understanding of KIAA0146 function in normal physiology and disease states.

How can researchers effectively analyze KIAA0146 interactions with the RAD51 recombination machinery?

Analyzing KIAA0146/SPIDR interactions with RAD51 and associated recombination proteins requires sophisticated methodological approaches. Researchers should implement a multi-tiered strategy beginning with co-immunoprecipitation assays to verify interactions in mouse cell lysates, followed by proximity ligation assays to visualize these interactions within cellular contexts. For quantitative binding assessments, surface plasmon resonance or microscale thermophoresis using purified recombinant proteins provides precise affinity measurements. The functional significance of these interactions can be evaluated through RAD51 focus formation assays following DNA damage induction, comparing wild-type cells to those expressing mutant KIAA0146 variants (such as truncation mutants analogous to the W280* variant identified in POI patients) . More advanced techniques include chromatin immunoprecipitation sequencing (ChIP-seq) to map genome-wide co-localization of KIAA0146 and RAD51, and single-molecule fluorescence resonance energy transfer (smFRET) to observe real-time dynamics of these interactions. When interpreting results, researchers should consider that KIAA0146 may function as part of larger protein complexes, potentially displaying different interaction properties in isolation versus cellular environments.

What methodologies are recommended for studying the role of KIAA0146 in reproductive disorders?

Investigating KIAA0146/SPIDR's role in reproductive disorders, particularly primary ovarian insufficiency (POI), requires specialized methodological approaches. Researchers should develop mouse models carrying specific SPIDR mutations identified in POI patients, such as the premature stop codons at positions W280*, R272*, and Q668* documented in clinical studies . These models should undergo comprehensive reproductive phenotyping, including histological analysis of ovarian follicle development, hormone profiling (FSH, LH, estradiol), fertility assessment, and age-related changes in ovarian reserve. At the cellular level, investigators should examine meiotic progression in oocytes from these models, with particular attention to DNA repair processes during prophase I where homologous recombination is essential. Single-cell transcriptomics of oocytes and granulosa cells can identify dysregulated pathways resulting from SPIDR deficiency. For translational relevance, researchers should establish collaborations with reproductive endocrinologists to obtain patient samples for comparative studies. When analyzing data, it is crucial to consider the complex interplay between DNA repair defects, cellular stress responses, and hormonal regulation that collectively contribute to reproductive pathologies.

How can researchers distinguish between direct and indirect effects of KIAA0146 mutations on genome stability?

Distinguishing between direct and indirect effects of KIAA0146/SPIDR mutations on genome stability presents a significant methodological challenge. Researchers should implement a strategic experimental framework beginning with reconstitution experiments using purified components to identify direct biochemical activities of KIAA0146. In parallel, proximity-dependent biotin identification (BioID) or APEX-based proximity labeling can map the complete protein interactome of KIAA0146 in relevant cell types, revealing both direct binding partners and proteins in nearby complexes. To assess genome-wide effects, investigators should combine γH2AX ChIP-seq to map DNA damage sites with whole-genome sequencing to identify mutation patterns characteristic of specific repair pathway deficiencies. Cellular studies comparing acute KIAA0146 depletion (using auxin-inducible degron systems) versus chronic knockout models help separate immediate functional consequences from adaptive responses. When analyzing patient-derived cells with SPIDR mutations (such as the premature termination variants documented in POI cases), complementation with wild-type or structure-specific mutants can determine which protein interactions are critical for genome maintenance . This comprehensive approach helps researchers delineate the direct mechanical roles of KIAA0146 in DNA repair from secondary effects resulting from genomic instability.

What are the critical quality control parameters for recombinant mouse KIAA0146 protein production?

Production of high-quality recombinant mouse KIAA0146 protein requires rigorous quality control measures throughout the expression and purification process. Researchers should implement comprehensive validation protocols including SDS-PAGE analysis to verify protein size and purity (expecting minimal degradation products), mass spectrometry to confirm sequence integrity, and circular dichroism spectroscopy to assess proper protein folding. Functional validation through binding assays with known interaction partners (particularly RAD51, SWSAP1-SWS1, and BLM helicase) is essential, as SPIDR's scaffolding function depends on maintaining correct interaction surfaces . Thermal shift assays provide valuable information about protein stability under various buffer conditions. When expressing full-length KIAA0146, researchers often encounter solubility challenges due to its relatively large size and potential for aggregation. To address this, consider expression strategies using solubility tags (such as MBP or SUMO), optimization of expression temperature (typically lower temperatures improve folding), and careful buffer screening during purification. For structural studies or biochemical assays requiring highly pure preparations, researchers should perform additional chromatography steps (ion exchange, size exclusion) following initial affinity purification. Batch-to-batch consistency should be monitored through standardized activity assays appropriate to KIAA0146's role in DNA repair processes.

What are the recommended approaches for studying KIAA0146 mutations identified in human patients using mouse models?

Translating human KIAA0146/SPIDR mutations to mouse models requires careful methodological considerations. When modeling patient-specific mutations such as the premature stop codons (W280*, R272*, Q668*) identified in POI patients , researchers should first confirm conservation of these positions between human and mouse sequences. CRISPR/Cas9 genome editing offers precise introduction of equivalent mutations in mouse embryonic stem cells for subsequent development of germline models. Alternatively, conditional knockout approaches using tissue-specific Cre expression (particularly in ovarian tissue for POI studies) paired with mutant cDNA rescue constructs can distinguish tissue-specific phenotypes. Researchers should implement comprehensive phenotypic characterization focusing on both molecular endpoints (DNA repair efficiency, protein interaction networks, transcriptional changes) and physiological outcomes (reproductive parameters, hormone profiles, age-related changes). For deeper mechanistic insights, single-cell approaches examining follicle development and oocyte quality provide valuable information about cell-type specific effects of SPIDR mutations. When interpreting results, researchers should consider potential compensatory mechanisms in mouse models that might not be active in human patients, as well as species-specific differences in reproductive physiology. Collaborative studies integrating findings from mouse models with patient-derived cells offer the most translational value in understanding how KIAA0146 mutations contribute to human disease.

What controls should be included when assessing KIAA0146 function in DNA repair assays?

Designing robust DNA repair assays to assess KIAA0146/SPIDR function requires comprehensive control strategies. Researchers should include multiple control conditions: wild-type cells/proteins as positive controls, complete knockout/depletion as negative controls, and rescue experiments with wild-type KIAA0146 to confirm phenotype specificity. When studying specific mutations (such as the W280*, R272*, or Q668* variants identified in POI patients) , researchers should include both structure-based mutants affecting specific domains and truncation mutants to distinguish domain-specific from complete loss-of-function effects. For DNA damage response assays, include technical controls for damage induction efficiency (e.g., γH2AX immunostaining). Time-course experiments are essential to distinguish defects in repair kinetics from complete repair failure. When analyzing protein interactions, implement specificity controls with unrelated proteins of similar size/charge characteristics. For genetic models, littermate controls are preferred to minimize background effects, and heterozygous animals should be analyzed to identify potential dominant-negative effects. In cellular systems, complementary approaches using both siRNA-mediated knockdown (acute depletion) and CRISPR knockout (complete absence) help distinguish between immediate functional requirements and compensatory adaptations. This comprehensive control strategy enables confident attribution of observed phenotypes to KIAA0146/SPIDR function rather than experimental artifacts or secondary effects.

How can researchers optimize immunoprecipitation protocols for studying KIAA0146 protein complexes?

Optimizing immunoprecipitation (IP) protocols for KIAA0146/SPIDR protein complexes requires careful consideration of experimental parameters to preserve physiologically relevant interactions. Researchers should employ a dual-tagging strategy, using both antibodies against endogenous SPIDR and epitope-tagged versions (such as FLAG or HA) to cross-validate findings. Cell lysis conditions significantly impact complex stability—start with gentle non-ionic detergents (0.5% NP-40 or 0.1% Triton X-100) in physiological salt concentrations, and systematically test modifications based on interaction strength. For DNA-mediated or chromatin-associated complexes, include DNase I or benzonase treatment conditions to distinguish direct protein-protein interactions from DNA-bridged associations. Crosslinking approaches (formaldehyde or DSS) can capture transient interactions, particularly important when studying dynamic DNA repair complexes involving RAD51, SWSAP1-SWS1, and BLM helicase . For challenging interactions, proximity-based approaches (BioID, APEX) provide complementary data. When analyzing results, compare KIAA0146 interactomes under both basal conditions and following DNA damage induction (e.g., ionizing radiation, mitomycin C) to identify damage-specific interactions. Mass spectrometry analysis should include quantitative approaches (SILAC, TMT) to distinguish specific interactors from background contaminants. This optimization strategy enables comprehensive characterization of KIAA0146 protein complexes under physiologically relevant conditions.

How should researchers interpret phenotypic variations in KIAA0146 mouse models compared to human patient data?

Interpreting phenotypic variations between KIAA0146/SPIDR mouse models and human patient data requires careful consideration of species-specific differences and methodological limitations. Researchers should develop a systematic comparison framework that maps equivalent phenotypic endpoints across species while accounting for physiological differences. For reproductive phenotypes associated with SPIDR mutations (such as POI in humans), consider that mouse reproductive physiology differs in aspects like follicle recruitment patterns and reproductive lifespan . When human patients with SPIDR mutations (such as W280*, R272*, Q668*) present with specific clinical features, researchers should establish analogous quantitative measurements in mouse models, such as follicle counts, hormone profiles, and fertility parameters. Phenotypic severity differences may result from species-specific genetic modifiers, requiring analysis of strain background effects in mouse models. Environmental factors affecting human patients but controlled in laboratory mice (such as environmental exposures or lifestyle factors) should be documented when comparing datasets. Statistical approaches should include power calculations based on expected effect sizes derived from human studies, and when possible, implement longitudinal studies in mouse models to capture age-related phenotype progression. This balanced interpretive approach acknowledges both the translational value and inherent limitations of mouse models in understanding human SPIDR-related pathologies.