Recombinant Mouse Motile sperm domain-containing protein 3 (Mospd3)

What is Mouse Mospd3 and what is its genetic structure?

Mouse Mospd3 (motile sperm domain containing 3) is a multi-pass membrane protein characterized by the presence of a major sperm protein (MSP) domain. The gene encoding Mospd3 is located in the mouse genome with several synonyms including 1190005J19Rik, 5133401H10Rik, Gtig2, and R124. The gene has an ORF size of 708 bp with RefSeq number BC003880 and UniGene ID Mm.41355. The gene has an ID of 68929 in genomic databases .

Structurally, Mospd3 belongs to the family of membrane-associated proteins containing MSP domains, which are evolutionarily conserved structural elements originally identified in nematode sperm proteins but now recognized in various multicellular organisms.

What is known about the function of Mospd3 in mouse development?

Mospd3 plays a critical role in cardiac development in mice. Studies have demonstrated that deletion of the Mospd3 gene is associated with defective cardiac development and neonatal lethality . This indicates that Mospd3 is essential for proper heart formation during embryonic development, although the precise molecular mechanisms remain to be fully elucidated.

The protein likely functions in membrane organization and cellular signaling pathways during organogenesis, particularly in cardiovascular tissues. Alternative splicing produces different isoforms that may have tissue-specific functions during development.

How does Mospd3 relate to other MOSPD family proteins?

The MOSPD family includes several proteins containing the motile sperm domain, with MOSPD1 and MOSPD3 being well-studied members. While Mospd3 is primarily associated with cardiac development, MOSPD1 has been identified as a direct target of the Wnt/β-catenin signaling pathway and shows elevated expression in colorectal cancer tissues compared to non-cancerous tissues .

Unlike Mospd3, MOSPD1 appears to be regulated through specific TCF-binding motifs in its 3′-flanking region that interact with transcription factor 7 like 2 (TCF7L2) and β-catenin . This differential regulation suggests distinct functions for these related proteins in development and disease.

What vectors are available for overexpressing mouse Mospd3 in experimental models?

Adenoviral vectors are available for overexpressing mouse Mospd3 in experimental models. Specifically, a human adenovirus Type 5 (dE1/E3) backbone expressing mouse Mospd3 under the CMV promoter (Ad-m-MOSPD3, catalog number ADV-264810) can be utilized for such studies .

These vectors can include optional reporter genes such as GFP, CFP, YFP, RFP, or mCherry to facilitate tracking of transduction efficiency. The vector is typically stored in DMEM with 2% BSA and 2.5% glycerol for stability .

When designing experiments using these vectors, researchers should consider including appropriate controls such as:

What are the recommended experimental approaches for studying Mospd3 function in cardiac development?

To study Mospd3 function in cardiac development, a multi-faceted experimental approach is recommended:

• Genetic Manipulation: Generate conditional knockout or knockdown models using Cre-loxP systems or CRISPR-Cas9 technology to target Mospd3 expression in cardiac tissues at specific developmental stages.

• Adenoviral Overexpression: Utilize adenoviral vectors expressing mouse Mospd3 (such as ADV-264810) for gain-of-function studies in cardiac cells or tissues .

• Histological Analysis: Perform detailed histological examination of cardiac tissues in Mospd3-deficient models to characterize developmental abnormalities.

• Molecular Profiling: Conduct transcriptomic and proteomic analyses of cardiac tissues with altered Mospd3 expression to identify downstream pathways and effectors.

• Functional Assays: Employ cardiomyocyte differentiation assays, contractility measurements, and electrophysiological analyses to assess the functional consequences of Mospd3 manipulation.

For in vivo studies, it's essential to control for genetic background, as mouse genetic reference panels (GRPs) show that strain differences can significantly impact cardiac phenotypes .

How should researchers design controlled experiments to study Mospd3's role in development?

When designing controlled experiments to study Mospd3's role in development, researchers should follow a systematic approach:

• Define variables precisely: Clearly identify independent variables (e.g., Mospd3 expression levels), dependent variables (e.g., cardiac developmental parameters), and potential confounding variables .

• Formulate specific hypotheses: For example, "Conditional deletion of Mospd3 in cardiac progenitor cells will lead to specific morphological defects in ventricular development."

• Design appropriate treatments: Consider using:

  • Gene knockout/knockdown approaches

  • Overexpression systems with adenoviral vectors like Ad-m-MOSPD3

  • Rescue experiments with wild-type and mutant Mospd3 constructs

• Group assignment: For mouse studies, use either:

  • Between-subjects design: Different treatment groups with matched genetic backgrounds

  • Within-subjects design: Comparing different tissues or developmental stages within the same animal

• Measurement protocols: Develop robust protocols for assessing:

  • Gene/protein expression levels

  • Cardiac morphology and function

  • Molecular interactions

  • Developmental outcomes

Control for environmental variables by standardizing housing conditions, diet, and handling procedures to minimize non-genetic sources of variation .

What are the optimal approaches for detecting and quantifying mouse Mospd3 protein in tissue samples?

For detecting and quantifying mouse Mospd3 protein in tissue samples, researchers should consider multiple complementary approaches:

• Immunohistochemistry/Immunofluorescence:

  • Use specific antibodies against mouse Mospd3

  • Polyclonal antibodies are available that recognize specific epitopes of the protein

  • Include appropriate negative controls (Mospd3-deficient tissues) and positive controls

  • Use confocal microscopy for subcellular localization studies

• Western Blotting:

  • Optimize protein extraction protocols for membrane proteins

  • Use reducing conditions appropriate for multi-pass membrane proteins

  • Validate antibody specificity using recombinant protein and knockout samples

  • Quantify bands using appropriate normalization controls

• ELISA/Protein Arrays:

  • Develop sandwich ELISA with capture and detection antibodies

  • Validate assay using recombinant protein standard curves

• Mass Spectrometry:

  • Use targeted proteomics approaches for absolute quantification

  • Develop specific peptide signatures for Mospd3 detection

  • Consider proximity labeling methods to identify interaction partners

When using antibodies, validate their specificity across different mouse strains, as genetic variation may affect epitope recognition. Commercial antibodies should be validated for the specific application intended .

How can systems genetics approaches be applied to understand Mospd3's role in cardiometabolic disease?

Systems genetics approaches provide powerful frameworks for understanding Mospd3's role in cardiometabolic disease:

• Genetic Reference Panels (GRPs): Utilize mouse GRPs such as the BXD lines, Collaborative Cross (CC), Diversity Outbred (DO), or Hybrid Mouse Diversity Panel (HMDP) to study natural genetic variation in Mospd3 and its impact on cardiometabolic traits .

• Integration of Multi-omics Data: Combine:

  • Genomics: Map quantitative trait loci (QTLs) affecting Mospd3 expression

  • Transcriptomics: Identify co-expression networks involving Mospd3

  • Proteomics: Characterize protein interactions and post-translational modifications

  • Metabolomics: Link Mospd3 variation to metabolic pathways

• Cross-species Analysis: Compare findings between mouse models and human datasets to identify conserved mechanisms, as the total genetic diversity across mouse strains (~71 million segregating SNPs) is comparable to human populations (~84.7 million SNPs) .

• Environmental Perturbations: Subject mouse GRPs to specific perturbations such as:

  • High-fat diets or other dietary challenges

  • Exercise interventions

  • Pharmacological treatments

  • Pathological insults mimicking human disease scenarios

• Network Analysis: Construct and analyze gene regulatory networks to:

  • Identify upstream regulators of Mospd3

  • Map downstream effectors

  • Discover pathway interconnections

  • Predict potential therapeutic targets

This integrative approach allows researchers to understand how genetic variation in Mospd3 contributes to cardiometabolic traits in the context of different genetic backgrounds and environmental conditions.

What are the recommended protocols for studying Mospd3 gene regulation?

For studying Mospd3 gene regulation, researchers should employ several complementary molecular biology techniques:

• Chromatin Immunoprecipitation (ChIP):

  • Use to identify transcription factors binding to the Mospd3 promoter or enhancer regions

  • Follow protocols similar to those used for MOSPD1 studies, where ChIP-qPCR identified TCF7L2 binding sites

  • Consider ChIP-seq to generate genome-wide binding profiles

• Reporter Assays:

  • Clone putative regulatory regions of Mospd3 into reporter vectors (e.g., pGL4.23)

  • Test enhancer/promoter activity in relevant cell types

  • Use site-directed mutagenesis to validate specific binding motifs

  • Normalize firefly luciferase activity to Renilla luciferase activity

• Chromosome Conformation Capture (3C):

  • Investigate long-range interactions between Mospd3 promoter and distant regulatory elements

  • Follow protocols involving cross-linking, restriction digestion (e.g., with HindIII), ligation, and nested PCR

• CRISPR-based Approaches:

  • Use CRISPR interference (CRISPRi) to inhibit specific regulatory elements

  • Apply CRISPR activation (CRISPRa) to enhance expression from specific promoters/enhancers

  • Use CRISPR-mediated deletion to remove regulatory elements

• Transcription Factor Binding Site Analysis:

  • Use databases like JASPAR to identify putative binding motifs in Mospd3 regulatory regions

  • Validate predictions with electrophoretic mobility shift assays (EMSA) and reporter assays

For all regulatory studies, consider the impact of cell type and developmental stage on regulatory mechanisms, as these can vary significantly.

What methodologies are recommended for investigating Mospd3 interaction partners?

To investigate Mospd3 interaction partners, researchers should employ multiple complementary approaches:

• Co-immunoprecipitation (Co-IP):

  • Use specific antibodies against Mospd3 to pull down protein complexes

  • Verify interactions with reciprocal Co-IP

  • Include appropriate controls (IgG, lysates from Mospd3-deficient cells)

• Proximity Labeling Methods:

  • BioID: Fuse Mospd3 to a biotin ligase to biotinylate proximal proteins

  • APEX2: Fuse Mospd3 to an engineered peroxidase for proximity labeling

  • These methods are particularly valuable for studying membrane protein interactions

• Yeast Two-Hybrid Screening:

  • Use the MSP domain or other functional domains as bait

  • Screen against cardiac or developmental cDNA libraries

  • Validate interactions with additional methods

• Mammalian Two-Hybrid Systems:

  • More physiologically relevant than yeast systems

  • Can be conducted in cardiac-relevant cell lines

• Mass Spectrometry-Based Approaches:

  • Affinity purification coupled with mass spectrometry (AP-MS)

  • Cross-linking mass spectrometry (XL-MS) to capture transient interactions

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interaction surfaces

• Fluorescence-Based Methods:

  • Förster resonance energy transfer (FRET)

  • Bimolecular fluorescence complementation (BiFC)

  • Fluorescence correlation spectroscopy (FCS)

When publishing interaction data, report detailed methodological parameters and include statistical analysis of replicate experiments to ensure reproducibility.

How do mouse models of Mospd3 deficiency compare with human MOSPD3 variations?

Comparing mouse models of Mospd3 deficiency with human MOSPD3 variations requires careful consideration of several factors:

• Phenotypic Comparison:

  • Mouse Mospd3 deletion is associated with defective cardiac development and neonatal lethality

  • Human MOSPD3 variations may present with varying degrees of cardiac abnormalities

  • Phenotypic severity might differ due to species-specific compensatory mechanisms

• Genetic Conservation:

  • Consider sequence homology between mouse Mospd3 and human MOSPD3

  • Antibody studies suggest approximately 88-89% sequence identity between mouse and human orthologs

  • Functional domains, particularly the MSP domain, are highly conserved

• Expression Patterns:

  • Compare tissue-specific expression patterns between species

  • Evaluate temporal expression during development

  • Assess alternative splicing patterns that may differ between species

• Methodological Approaches:

  • Generate humanized mouse models expressing human MOSPD3 variants

  • Use patient-derived iPSCs differentiated into cardiomyocytes to verify findings from mouse models

  • Apply systems genetics approaches to link human genetic variation to cardiac phenotypes

• Regulatory Considerations:

  • Compare transcriptional regulation mechanisms between species

  • Evaluate whether pathways like Wnt/β-catenin signaling (which regulates MOSPD1 ) also affect MOSPD3 in both species

When designing studies, it's important to recognize that while mouse genetic diversity (~71 million segregating SNPs) is comparable to human populations (~84.7 million SNPs) , species-specific differences in development and physiology must be considered when translating findings.

What are the current challenges in integrating mouse Mospd3 research findings with human cardiovascular disease studies?

The integration of mouse Mospd3 research with human cardiovascular disease studies faces several key challenges:

• Species-Specific Differences:

  • Developmental timing differences between mouse and human cardiac development

  • Different compensatory mechanisms may exist when Mospd3/MOSPD3 is disrupted

  • Cardiac physiology differences (e.g., heart rate, metabolic requirements)

• Genetic Background Effects:

  • Phenotypic outcomes of Mospd3 manipulation may vary across different mouse strains

  • Human genetic diversity creates additional complexity in translating mouse findings

  • Controlled mouse genetic reference panels offer advantages but also limitations in modeling human populations

• Technical and Methodological Barriers:

  • Limited availability of well-validated antibodies and reagents that work across species

  • Challenges in generating equivalent genetic modifications in mouse and human models

  • Differences in experimental conditions and protocols between labs

• Data Integration Challenges:

  • Combining heterogeneous data types from different experimental platforms

  • Harmonizing mouse and human genomic coordinates and annotations

  • Integrating multi-omics data across species to identify conserved mechanisms

• Translational Relevance Assessment:

  • Determining which aspects of mouse Mospd3 biology are most relevant to human disease

  • Identifying human MOSPD3 variants of clinical significance

  • Developing appropriate functional assays to test variant effects

To address these challenges, researchers should:

• Use complementary human and mouse discovery platforms

• Apply systems genetics approaches to reveal conserved gene-trait associations

• Validate findings across multiple mouse strains and human populations

• Develop standardized protocols for cross-species data integration and analysis