Recombinant Danio rerio Transmembrane protein 71 (tmem71)

What are the optimal storage and handling conditions for recombinant TMEM71 protein?

For optimal stability and activity of recombinant Danio rerio TMEM71:

• Store at -20°C for regular use

• For extended storage, maintain at -80°C

• Avoid repeated freeze-thaw cycles which can lead to protein degradation

• Working aliquots may be stored at 4°C for up to one week

• The protein is typically supplied in a Tris-based buffer containing 50% glycerol, specifically optimized for TMEM71 stability

How can I access zebrafish TMEM71 resources for my research?

Several database resources provide valuable information for TMEM71 research:

• ZFIN (Zebrafish Information Network): The central repository for zebrafish genetic, genomic, and developmental data. ZFIN provides:

  • Gene expression patterns

  • Mutant phenotypes

  • Protein domain information with links to InterPro

  • Available antibodies and reagents

• Alliance of Genome Resources: Integrates ZFIN data with other model organism databases, providing:

  • Cross-species orthology information

  • Gene and allele pages

  • AllianceMine data mining tools for querying gene, orthology, allele, and ontology data

• UniProt: Contains protein sequence and annotation data (UniProt accession: B0S728)

What methods are commonly used for detecting TMEM71 expression in zebrafish?

Detection methods include:

• RNA expression analysis:

  • RT-PCR for targeted detection

  • RNA-Seq or microarray for genome-wide expression profiling

  • In situ hybridization for spatial expression patterns during development

• Protein detection:

  • Immunohistochemistry using specific antibodies

  • Western blotting for protein expression levels

  • Fluorescent reporter systems: transgenic fish expressing TMEM71 fused to fluorescent proteins like eGFP or mCherry

How can Perturb-seq technology be applied to study TMEM71 function in zebrafish?

Perturb-seq combines CRISPR-based perturbations with single-cell RNA sequencing to comprehensively characterize gene function. For TMEM71 studies:

Methodology workflow:

• Design phase:

  • Generate CRISPR guide RNAs targeting TMEM71

  • Consider the IterPert approach for selecting optimal perturbations

• Sequential experimental design:

  • Conduct initial perturbation experiments targeting TMEM71

  • Train machine learning models on the resulting data

  • Use these models to strategically select subsequent perturbations

  • Repeat this cycle to efficiently explore the function of TMEM71 and its interaction network

• Analysis considerations:

  • Set up proper experimental controls (non-targeting guides)

  • Consider batch sizes of approximately 100 perturbations per round

  • Optimize for approximately 5 experimental rounds

This sequential approach is particularly valuable for TMEM71 as it allows researchers to explore both the direct effects of TMEM71 perturbation and its interaction with other genes, while minimizing the number of experiments required.

What signaling pathways are associated with TMEM71 and how can they be studied in zebrafish?

Based on studies primarily in human systems, TMEM71 shows significant associations with several key signaling pathways:

Methodological approaches for zebrafish studies:

• Pharmacological studies: Treat zebrafish embryos with pathway-specific inhibitors (e.g., PI3K inhibitors) and analyze effects on TMEM71 expression or TMEM71-related phenotypes

• Genetic interaction studies: Generate double mutants of TMEM71 and key components of these signaling pathways

• Reporter assays: Use transgenic zebrafish lines with pathway-specific reporters to visualize pathway activity in TMEM71 mutant backgrounds

• Biochemical analyses: Assess phosphorylation states of key pathway components in wild-type versus TMEM71-deficient zebrafish

How does TMEM71 relate to immune function based on current research?

Research on human TMEM71 indicates significant immunological associations that could be explored in zebrafish models:

• Immune checkpoint correlations:

  • TMEM71 shows high positive correlation with PD-1 and PD-L1 expression

  • Strong associations with TIM-3 and B7-H3 immune checkpoints

• Inflammatory activities:

  • Positively correlated with HCK, LCK, MHC-II, and STAT1 inflammatory markers

  • Negatively correlated with IgG and interferon signaling

• Functional implications:

  • May play a role in macrophage activation

  • Potentially involved in T-cell signaling transduction

Zebrafish research approaches:

• Analyze immune cell populations in TMEM71-deficient zebrafish larvae

• Challenge with pathogens to assess immune response efficacy

• Perform transcriptome analysis focusing on immune-related genes

• Use fluorescent reporter lines marking immune cell populations to track cellular responses

What are the most effective approaches for studying TMEM71 localization in zebrafish tissues?

To effectively visualize and analyze TMEM71 localization:

• Transgenic fluorescent reporter systems:

  • Generate transgenic lines expressing TMEM71-fluorescent protein fusions

  • Use tissue-specific promoters to study cell-type specific localization

  • Combine with membrane markers to assess precise membrane localization

• Live imaging protocol:

  • Use 72-96 hours post-fertilization (hpf) hatched larvae

  • Embed in 0.3% agar containing PTU (to suppress pigmentation) and tricaine (0.01%) for anesthesia

  • Perform confocal microscopy at room temperature

• Colocalization studies:

  • Combine TMEM71 visualization with markers for specific subcellular compartments

  • Use double transgenic lines expressing different fluorescent markers

  • Apply quantitative colocalization analysis techniques

• High-resolution approaches:

  • Super-resolution microscopy for nanoscale localization

  • Electron microscopy with immunogold labeling for ultrastructural localization

How can CRISPR/Cas9 techniques be optimized for TMEM71 functional studies?

CRISPR/Cas9 optimization for TMEM71 studies:

• Guide RNA design considerations:

  • Target early exons to maximize functional disruption

  • Use zebrafish-specific design tools to minimize off-target effects

  • Consider multiple guides targeting different regions to address potential compensation

• Delivery protocol:

  • Microinject CRISPR/Cas9 components at one-cell stage embryos

  • Typical mixture: 300 ng/μL Cas9 mRNA and 25 ng/μL sgRNA

• Validation strategies:

  • T7 endonuclease assay or direct sequencing to confirm mutations

  • RT-PCR and Western blotting to verify reduced expression

  • Phenotypic analysis based on predicted TMEM71 function

• Advanced applications:

  • Consider tissue-specific or inducible CRISPR systems for temporal control

  • Use homology-directed repair for precise mutations or tagging

What are the considerations for developing disease models related to TMEM71 in zebrafish?

Clinical research has indicated that TMEM71 expression is associated with survival outcomes in human glioma patients, with a hazard ratio (HR) of 7.035 (95% CI: 1.571-31.494) in univariate analysis and 18.43 (95% CI: 2.463-138.02) in multivariate analysis .

Zebrafish disease modeling approaches:

• Genetic models:

  • Generate TMEM71 knockout or overexpression models

  • Create precise mutations that mimic human disease variants

  • Assess pathological phenotypes and compare to human disease manifestations

• Functional assessment:

  • Perform behavioral assays if neurological functions are affected

  • Analyze histological changes in relevant tissues

  • Measure pathway activities associated with disease progression

• Drug screening applications:

  • Use zebrafish larvae for high-throughput screening of compounds that modulate TMEM71-related phenotypes

  • Apply the in vivo vertebrate model advantages of zebrafish for assessing drug effects

• Translational applications:

  • Link findings to human disease data through orthology relationships

  • Utilize resources like the Alliance Disease page for cross-species comparisons

What methods should be used to validate antibodies against zebrafish TMEM71?

A comprehensive antibody validation strategy includes:

• Specificity testing:

  • Western blot analysis using wild-type and TMEM71 knockout/knockdown tissues

  • Preabsorption with immunizing peptide to confirm specificity

  • Mass spectrometry verification of immunoprecipitated proteins

• Technical validation:

  • Test different fixation and antigen retrieval methods for immunohistochemistry

  • Validate across multiple applications (Western blot, IHC, IP)

  • Compare results from multiple antibodies targeting different epitopes

• Biological validation:

  • Compare antibody staining patterns with mRNA expression data

  • Use transgenic lines expressing tagged TMEM71 as positive controls

  • Verify subcellular localization is consistent with predicted transmembrane topology

• Documentation and transparency:

  • Record detailed validation protocols and results

  • Report negative results to prevent others from using non-specific antibodies

  • Check ZFIN for existing validated antibodies against zebrafish TMEM71

How can contradictory data about TMEM71 function be reconciled across different experimental systems?

To resolve contradictory findings in TMEM71 research:

• Systematic approach to experimental design:

  • Apply the sequential optimal experimental design principles described for Perturb-seq

  • Strategically select experimental conditions that can resolve contradictions

  • Use machine learning models to predict the most informative next experiments

• Multi-level validation:

  • Test hypotheses across multiple model systems (cell lines, zebrafish, mouse)

  • Compare results from different perturbation approaches (CRISPR knockout, morpholino knockdown, overexpression)

  • Validate with complementary techniques (transcriptomics, proteomics, functional assays)

• Contextual analysis:

  • Consider developmental stage-specific effects

  • Analyze tissue-specific or cell type-specific functions

  • Examine genetic background influences

• Data integration:

  • Utilize bioinformatics resources like ZFIN and AllianceMine to integrate findings

  • Apply systems biology approaches to model complex interactions

  • Develop computational models that can explain apparently contradictory results

What considerations should be made when designing expression systems for recombinant zebrafish TMEM71?

Designing effective expression systems for transmembrane proteins like TMEM71 requires special considerations:

• Expression system selection:

  • Mammalian cells: Provide proper folding and post-translational modifications

  • Insect cells: Balance between mammalian-like processing and higher yield

  • Bacterial systems: Higher yield but may require refolding for proper structure

• Vector design considerations:

  • Include appropriate purification tags (His, GST, FLAG)

  • Consider fusion partners to improve solubility

  • Include protease cleavage sites for tag removal

  • Optimize codon usage for the expression system

• Membrane protein-specific strategies:

  • Consider detergent screening for optimal solubilization

  • Test nanodiscs or liposomes for functional reconstitution

  • Evaluate truncation constructs to identify stable domains

• Quality control metrics:

  • Size exclusion chromatography to verify monodispersity

  • Circular dichroism to confirm secondary structure

  • Functional assays specific to predicted activities

How might single-cell RNA sequencing advance our understanding of TMEM71 function?

Single-cell RNA sequencing offers powerful approaches to understand TMEM71 biology:

• Cell type-specific expression analysis:

  • Identify cell populations that express TMEM71 at different developmental stages

  • Correlate with cell type-specific markers to define expression patterns

  • Map temporal dynamics of expression during development

• Perturbation analysis:

  • Apply Perturb-seq methodology as described earlier

  • Analyze cell type-specific responses to TMEM71 perturbation

  • Identify differential effects across developmental lineages

• Regulatory network inference:

  • Identify genes co-regulated with TMEM71

  • Map upstream regulators and downstream targets

  • Build gene regulatory networks centered on TMEM71

• Clinical applications:

  • Compare expression patterns in disease models

  • Identify cell populations with altered TMEM71 expression in pathological states

  • Discover potential therapeutic targets in TMEM71-related pathways

What is the potential role of TMEM71 in zebrafish models of human disease?

Based on human TMEM71 associations with disease processes, zebrafish models offer opportunities to investigate:

• Oncology applications:

  • Human TMEM71 shows significant associations with glioma progression and survival

  • Zebrafish cancer models could elucidate mechanisms of TMEM71 in tumor development

  • Screen for compounds that modulate TMEM71-related cancer phenotypes

• Immunological disease modeling:

  • Given TMEM71's connections to immune checkpoint molecules (PD-1, PD-L1, TIM-3)

  • Investigate roles in autoimmune conditions or immunodeficiencies

  • Test immunomodulatory approaches targeting TMEM71-related pathways

• Developmental disorder investigation:

  • Study potential roles in neural development based on signaling pathway associations

  • Investigate phenotypes resulting from TMEM71 dysfunction during embryogenesis

  • Compare to human developmental disorders with similar genetic profiles

• Translational research pipeline:

  • Generate humanized zebrafish models expressing human TMEM71 variants

  • Use for high-throughput screening of therapeutic candidates

  • Validate findings in higher vertebrate models before clinical translation