Recombinant Danio rerio Abhydrolase domain-containing protein FAM108C1 (fam108c1)

What is Abhydrolase domain-containing protein FAM108C1 in Danio rerio?

Abhydrolase domain-containing protein FAM108C1 (fam108c1) is a member of the abhydrolase superfamily expressed in Danio rerio (zebrafish). It contains a characteristic alpha/beta hydrolase fold and is believed to play roles in lipid metabolism and cellular signaling pathways. The recombinant form of this protein can be produced in expression systems such as E. coli for research purposes . While the precise physiological function remains under investigation, its homologs in other species have been implicated in membrane lipid remodeling and metabolic processes.

What expression systems are suitable for producing Recombinant Danio rerio FAM108C1?

Recombinant Danio rerio FAM108C1 can be effectively produced in prokaryotic expression systems, with E. coli being the most common host organism . For standard research applications, bacterial expression offers advantages of high yield and cost-effectiveness. The protein is typically produced with affinity tags (such as His-tag or GST-tag) to facilitate purification. Alternative expression systems including yeast, insect cells, or mammalian cells may provide improved post-translational modifications when needed for specific functional studies, although these systems typically result in lower yields and higher production costs.

How can I validate the activity of recombinant FAM108C1 protein in vitro?

Validation of recombinant FAM108C1 activity can be performed through multiple complementary approaches:

• Enzymatic activity assays using synthetic substrates that contain ester or amide bonds

• Thermal shift assays to assess protein stability and ligand binding

• Western blotting with specific antibodies to confirm protein identity and integrity

• Mass spectrometry to verify protein sequence and post-translational modifications

• Size exclusion chromatography to assess oligomeric state and proper folding

Activity should be compared against appropriate positive and negative controls, including heat-inactivated protein samples and known substrates for abhydrolase domain-containing proteins.

What are appropriate storage conditions for maintaining FAM108C1 stability?

For optimal stability of Recombinant Danio rerio FAM108C1, the following storage conditions are recommended:

The protein should be maintained in a pH-buffered solution (typically pH 7.4-8.0) with stabilizing agents such as glycerol and potentially reducing agents if the protein contains critical cysteine residues . Avoid shipping without dry ice as temperature fluctuations can significantly reduce activity.

How does FAM108C1 function in zebrafish pronephric development and what are the implications for human nephrology research?

FAM108C1 has potential roles in zebrafish kidney development and function, though specific mechanisms remain under investigation. The zebrafish pronephric kidney shares significant structural and functional homology with mammalian nephrons, making it a valuable model for studying kidney development and disease .

When investigating FAM108C1 in kidney research:

• Expression patterns can be visualized in zebrafish larvae using fluorescently-tagged antibodies against FAM108C1 and confocal microscopy

• Morpholino knockdown or CRISPR/Cas9 gene editing can be used to assess phenotypic effects of FAM108C1 depletion

• Microinjection of modified mRNA can be employed for rescue experiments to confirm specificity

• Functional assays can measure pronephric filtration rates using fluorescent dextran clearance assays

Significantly, zebrafish larvae offer advantages for nephrotoxicity studies as demonstrated with gentamicin , potentially providing insights into how FAM108C1 may modulate nephrotoxic responses or contribute to kidney development.

What systems biology approaches can be used to characterize FAM108C1 function in zebrafish models?

Systems biology approaches provide comprehensive frameworks for characterizing FAM108C1 function:

• Multi-omics integration: Combine transcriptomics, proteomics, and metabolomics data from wild-type and FAM108C1-deficient zebrafish to construct functional networks

• Pathway analysis: Employ bioinformatic tools to identify enriched pathways and biological processes associated with FAM108C1 expression or depletion

• Protein-protein interaction mapping: Use techniques such as proximity labeling, co-immunoprecipitation combined with mass spectrometry, or yeast two-hybrid screening to identify direct interaction partners

• Computational modeling: Develop predictive models for FAM108C1 function based on sequence homology with better-characterized abhydrolase family members

• Cross-species validation: Compare findings in zebrafish with mammalian models to establish evolutionary conservation of function

Implementing these approaches requires careful experimental design with appropriate controls and statistical analyses to account for biological variability .

How can CRISPR/Cas9 gene editing be optimized for studying FAM108C1 function in zebrafish?

CRISPR/Cas9 gene editing provides powerful tools for studying FAM108C1 function through targeted genetic manipulation:

For optimal CRISPR/Cas9 editing efficiency:

• Design multiple guide RNAs targeting conserved functional domains of FAM108C1

• Validate editing efficiency using T7 endonuclease assays or direct sequencing

• Screen F0 mosaic founders for germline transmission

• Establish stable lines through careful breeding and genotyping

• Confirm knockout at protein level using specific antibodies

Remember that off-target effects must be controlled through careful guide RNA design and validation with rescue experiments using wild-type FAM108C1 mRNA microinjection.

What are the challenges in correlating in vitro enzymatic activity of FAM108C1 with its in vivo function in zebrafish models?

Several key challenges exist when attempting to correlate in vitro enzymatic characterization with in vivo function:

• Physiological substrate identification: The natural substrates of FAM108C1 in zebrafish may differ from synthetic substrates used in vitro, requiring unbiased metabolomic approaches to identify endogenous targets

• Cellular context: The protein may require specific cellular cofactors, membrane environments, or post-translational modifications not present in recombinant systems

• Developmental and tissue-specific regulation: Expression and activity of FAM108C1 likely varies across developmental stages and tissues, necessitating temporal and spatial resolution in analysis

• Redundant enzymatic functions: Other hydrolases may compensate for FAM108C1 deficiency in vivo, masking phenotypes observed in knockout models

• Technical limitations: Quantifying enzymatic activity in living zebrafish remains challenging and may require development of specific biomarkers or reporter systems

These challenges highlight the importance of integrating multiple experimental approaches, from biochemical characterization to in vivo functional studies, to develop a comprehensive understanding of FAM108C1 biology.

How should zebrafish models be designed for optimal study of FAM108C1 function?

When designing zebrafish models to study FAM108C1 function, consider these methodological approaches:

• Selection of developmental stages: Embryonic and larval stages (24-120 hours post-fertilization) offer advantages of transparency and rapid development for high-throughput screening

• Genetic background considerations: Use established wild-type strains (AB, TU, or WIK) with known genetic backgrounds to minimize variability

• Control groups: Include appropriate controls such as:

  • Wild-type siblings from the same clutch

  • Sham-injected controls for microinjection studies

  • Non-targeting guide RNA controls for CRISPR studies

  • Vehicle controls for drug treatment studies

• Sample size determination: Power analysis should be performed to determine appropriate sample sizes based on expected effect sizes

• Housing conditions standardization: Maintain consistent temperature (28.5°C), photoperiod (14h light/10h dark), and water quality parameters

• Ethical considerations: Design experiments following the 3Rs principles (Replacement, Reduction, Refinement) with appropriate ethical approvals

For imaging studies of zebrafish larvae, confocal microscopy with specific staining protocols allows visualization of pronephric structures to assess FAM108C1 localization and function within kidney tissues .

What are the most effective methods for assessing FAM108C1 expression and localization in zebrafish tissues?

Multiple complementary approaches can be used to assess FAM108C1 expression and localization:

• Quantitative PCR (qPCR): Measures mRNA expression levels across different tissues and developmental stages

  • Requires careful primer design specific to zebrafish FAM108C1

  • Normalized against stable reference genes (e.g., ef1α, rpl13a)

• In situ hybridization: Visualizes spatial expression patterns of FAM108C1 mRNA

  • Whole-mount techniques are effective for embryos and larvae

  • Section-based approaches for adult tissues

• Immunohistochemistry/Immunofluorescence: Determines protein localization at cellular and subcellular levels

  • May require generation of zebrafish-specific antibodies

  • Co-staining with organelle markers clarifies subcellular localization

• Transgenic reporter lines: Generation of FAM108C1-fluorescent protein fusions

  • Enables real-time visualization in living zebrafish

  • Can be combined with tissue-specific promoters for targeted expression

• Western blotting: Quantifies protein expression across different tissues

  • Requires careful sample preparation and loading controls

  • Can detect post-translational modifications with specific antibodies

For confocal imaging of kidney structures, techniques using fluorescent dextran conjugates can be employed to assess functional aspects alongside localization studies .

How can RNA-seq data be effectively analyzed to understand FAM108C1 regulatory networks in zebrafish?

RNA-seq analysis for understanding FAM108C1 regulatory networks should follow these methodological steps:

• Experimental design:

  • Compare wild-type vs. FAM108C1 knockout/knockdown models

  • Include biological replicates (minimum n=3 per condition)

  • Consider developmental time points and tissue specificity

• Quality control and preprocessing:

  • Assess read quality with tools like FastQC

  • Trim adapters and low-quality bases

  • Align to zebrafish reference genome (GRCz11/danRer11)

• Differential expression analysis:

  • Use established tools (DESeq2, edgeR) with appropriate statistical thresholds

  • Apply false discovery rate correction for multiple testing

  • Validate key findings with qPCR

• Network analysis:

  • Perform weighted gene co-expression network analysis (WGCNA)

  • Identify hub genes and modules associated with FAM108C1 function

  • Apply strict similarity thresholds to minimize false positives

• Functional enrichment:

  • Analyze enriched Gene Ontology terms and pathways

  • Compare with known abhydrolase domain protein functions

  • Cross-reference with human ortholog data when available

• Integration with other omics data:

  • Combine with proteomics and metabolomics when available

  • Develop predictive models for FAM108C1 regulatory networks

The analysis should distinguish between direct and indirect effects of FAM108C1 perturbation by incorporating time-course data when possible .

What considerations are important when designing drug screening assays using zebrafish models to identify compounds affecting FAM108C1 function?

When designing zebrafish-based drug screening assays targeting FAM108C1 function:

• Assay development:

  • Establish clear, quantifiable phenotypic readouts related to FAM108C1 function

  • Develop high-content imaging protocols for automated analysis

  • Optimize compound delivery methods (water exposure vs. microinjection)

• Screening logistics:

  • Determine appropriate developmental stages for treatment (typically 24-120 hpf)

  • Standardize embryo collection and dechorionation procedures

  • Implement appropriate positive and negative controls

• Dosing considerations:

  • Establish concentration ranges that balance efficacy and toxicity

  • Account for absorption, distribution, and metabolism differences between zebrafish and mammals

  • Consider exposure duration and timing relative to developmental events

• Validation strategies:

  • Confirm target engagement through direct binding assays

  • Perform dose-response studies to establish potency

  • Validate hits in secondary assays with different readouts

• Translational aspects:

  • Consider pharmacokinetic differences between zebrafish and mammals

  • Validate findings in mammalian cell culture or other model organisms

  • Assess structural similarities between zebrafish and human FAM108C1 orthologs

Zebrafish larvae provide an excellent model for nephrotoxicity studies, making them particularly valuable for screening compounds that might affect FAM108C1 function in kidney tissues .

What are the emerging trends in FAM108C1 research using zebrafish models?

Emerging research trends for FAM108C1 in zebrafish models include:

• Integration of CRISPR/Cas9 genome editing with high-throughput phenotypic screening

• Application of single-cell RNA sequencing to characterize cell type-specific roles

• Development of zebrafish reporter lines for real-time visualization of FAM108C1 activity

• Comparative studies between zebrafish and mammalian FAM108C1 orthologs

• Investigation of potential roles in disease models, particularly kidney-related disorders

• Systems biology approaches that integrate multi-omics data to build comprehensive functional networks

These developments are enabling more sophisticated understanding of FAM108C1 biology while leveraging the unique advantages of zebrafish as a vertebrate model system, including optical transparency, external development, and genetic tractability.

How can findings from zebrafish FAM108C1 studies be translated to human health applications?

Translating zebrafish FAM108C1 findings to human applications requires:

• Comparative genomics analysis:

  • Establish orthology relationships between zebrafish FAM108C1 and human counterparts

  • Compare protein domain structures and key functional residues

  • Assess conservation of regulatory elements and expression patterns

• Validation in mammalian models:

  • Confirm key findings in mammalian cell cultures

  • Validate in mouse models when appropriate

  • Consider human tissue samples or organoids for direct relevance

• Disease relevance assessment:

  • Explore associations with human conditions through database mining

  • Investigate potential biomarker applications

  • Consider therapeutic targeting strategies if disease associations are established

• Methodological considerations:

  • Account for species-specific differences in experimental design

  • Apply appropriate statistical methods for cross-species comparisons

  • Implement rigorous controls to validate translational relevance

The zebrafish model offers particularly strong translational potential for kidney-related studies due to the structural and functional similarities between zebrafish pronephros and human nephrons , potentially making FAM108C1 findings especially relevant to human nephrology research.

What are the current limitations in FAM108C1 research and how might they be addressed?

Current limitations in FAM108C1 research include:

• Limited functional characterization:

  • The physiological substrates remain largely unknown

  • Specific signaling pathways and interaction partners are not fully characterized

  • Solution: Apply unbiased substrate screening and interactome analysis

• Technical challenges:

  • Lack of highly specific antibodies for zebrafish FAM108C1

  • Challenges in measuring enzymatic activity in vivo

  • Solution: Develop improved tools including CRISPR knock-in tags and activity-based probes

• Knowledge gaps in regulatory mechanisms:

  • Transcriptional and post-translational regulation poorly understood

  • Tissue-specific functions not fully delineated

  • Solution: Apply systems biology approaches to construct regulatory networks

• Translational barriers:

  • Species differences may limit direct application to human health

  • Compensatory mechanisms in model systems can mask phenotypes

  • Solution: Validate findings across multiple model systems and human samples