Recombinant Danio rerio Transmembrane protein 151B (tmem151b)

What is TMEM151B and what is its structural characterization in zebrafish?

TMEM151B (Transmembrane protein 151B) is a multi-pass membrane protein in zebrafish (Danio rerio) consisting of 513 amino acids. Based on sequence analysis, zebrafish TMEM151B contains:

• A transmembrane domain structure

• Full amino acid sequence: MSPAAPVTESSAAEVHREQTDAPREPQRPLKQSLSKSLCRESHWKCLLLSLLMYCCVIAMTWCQVTKVTRLSFDSTFKVKSMIYHDSPCSNGYIYIPAFLVMLYV VYLVECWQCYSRNELQFKVDLESVTERVQRMQQATPCIWWKAISYHYIRRTRQVTRYRNGDAYTSTQVYHERVNTHVAEAEFDYGNCGVKDIPKHLAGSDGFPVTKLRFTKCFSFANVESENSYLTQRARFFTENEGLDDYMEAREGMHLKNVEFKEYMVAFADPNRLPWYASTCSFWLAAAFTLSWPLRVLTEYRTAYLHYHVEKLFGFDYVSVTPLDHERPFCRHIPRVNTIDSTELEWHIRSNQQLVPSYSEAVLMNLTQQSSCNTFSARGIGAAGGNGFGGYRQNCERCHRSISSSSIFSRSALSICNSSNPRLAFSSSRFSLGRLYGSRRSCLWQSRSSSLNDPGCPTESTRCLANEESPPSPPAYQDALYFPVLIIHRNEGCIAHDHHSLHRNGSCVETSL

The protein has been classified with several synonyms including zgc:171639 in zebrafish genomic databases . Its UniProt accession number is A7E2I7, and it represents a full-length protein with an expression region of 1-513 amino acids .

What expression systems are typically used for recombinant Danio rerio TMEM151B production?

Common expression systems for recombinant zebrafish TMEM151B production include:

Most commercial recombinant TMEM151B is produced using one of these systems, with the tag type determined during the production process to optimize for protein stability and function . The recombinant protein is typically maintained in a storage buffer containing Tris-based buffer with 50% glycerol to optimize stability .

What are the recommended storage and handling protocols for recombinant Danio rerio TMEM151B?

For optimal stability and activity of recombinant Danio rerio TMEM151B:

• Store at -20°C for routine storage

• For extended storage, maintain at -20°C or -80°C

• Avoid repeated freeze-thaw cycles which can compromise protein integrity

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

• The protein is typically supplied in a glycerol-containing buffer to enhance stability

When handling the recombinant protein, it is recommended to:

• Thaw aliquots rapidly at room temperature

• Keep on ice during experimental procedures

• Use sterile techniques to prevent contamination

• Document freezing/thawing cycles to monitor potential degradation

What gene-editing approaches are most effective for studying tmem151b function in zebrafish?

Several gene-editing approaches have been validated for studying zebrafish genes like tmem151b:

CRISPR-Cas9 System:
The CRISPR-Cas9 system has been successfully used to generate knockout zebrafish lines for multiple transmembrane proteins. For tmem151b:

• Design guide RNAs targeting early exons to ensure complete loss of function

• Inject a mixture of Cas9 mRNA (100-300 pg) and guide RNA (25-50 pg) into one-cell stage embryos

• Identify potential founders by sequencing PCR products from F0 embryo genomic DNA

• Establish stable lines through outcrossing to wild-type strains

• Confirm mutations through DNA sequencing and verify frameshift mutations that result in truncated proteins

Morpholino-Modified Antisense Oligonucleotides:
For transient knockdown studies, morpholinos remain valuable:

• Design morpholinos targeting either the start site or splice sites of tmem151b

• Dissolve morpholinos in sterile water to create a 1 mM stock solution

• For working solutions, dilute to approximately 0.18 mM with Danieau's water

• Microinject 1-2 nl into one-cell stage embryos using calibrated needles

• Include standard control morpholinos in parallel experiments

• Validate knockdown efficiency through RT-PCR, Western blotting, or in vitro translation assays

When using morpholinos, it's critical to verify specificity by:

• Using a second non-overlapping morpholino

• Performing rescue experiments with co-injection of capped mRNA

• Including appropriate controls to rule out off-target effects

How can transgenic zebrafish lines be generated to study TMEM151B expression and function?

For generating transgenic zebrafish lines to study TMEM151B, the Tol2 transposon system has proven highly effective:

Tol2 Transposon-Based Transgenesis Protocol:

• Construct Design:

  • Create a donor plasmid containing the tmem151b gene or regulatory elements flanked by Tol2 transposon repeats

  • Consider fusion with fluorescent reporters (GFP, RFP) for visualization

  • Include tissue-specific or inducible promoters as needed

• Microinjection:

  • Co-inject the Tol2 construct (20-40 pg) and Tol2 transposase mRNA (20-30 pg) into fertilized eggs at the one-cell stage

  • The transposase facilitates removal of the Tol2 construct from the donor plasmid and incorporation into the genome

• Founder Identification:

  • Screen injected embryos for reporter expression

  • Raise positive embryos to adulthood

  • Outcross potential founders to wild-type fish

  • Identify germline transmission by screening F1 offspring

• Line Maintenance:

  • Establish stable lines through successive generations

  • Consider using the optimized pIGLET system for targeted insertions at validated genomic loci

For more sophisticated analysis, consider combining with other genetic tools:

• The Gal4-UAS system for cell-type-specific expression

• The Cre-lox system for conditional gene manipulation

• The FRET system for studying protein interactions in vivo

These transgenic approaches allow for spatiotemporal analysis of TMEM151B expression and function throughout zebrafish development.

What methods are most effective for analyzing TMEM151B expression patterns in zebrafish?

Multiple complementary approaches can be used to analyze TMEM151B expression:

RNA-Based Methods:

• In situ hybridization:

  • Generate RNA probes targeting tmem151b mRNA

  • Use chromogenic or fluorescent detection for tissue localization

  • Compatible with whole-mount preparation for embryos/larvae

• RT-qPCR:

  • Extract RNA from specific tissues or developmental stages

  • Design primers spanning exon-exon junctions

  • Normalize to validated reference genes for zebrafish

Protein-Based Methods:

• Immunohistochemistry/Immunofluorescence:

  • Use anti-TMEM151B antibodies for protein localization

  • Apply at dilutions of 1:50-1:200 for optimal results

  • Combine with neuronal or other markers for co-localization studies

• Western Blotting:

  • Extract proteins from zebrafish tissues

  • Separate proteins by SDS-PAGE

  • Use specific antibodies at recommended dilutions

Reporter-Based Methods:

• Transgenic Reporter Lines:

  • Generate lines with fluorescent proteins driven by the tmem151b promoter

  • Image using confocal microscopy for cellular resolution

  • Combine with calcium indicators for functional studies

For quantitative temporal expression analysis, integrate RNA-seq and proteomic approaches to understand both transcriptional and translational regulation of TMEM151B during zebrafish development .

What are the potential roles of TMEM151B in zebrafish development and disease models?

While specific functions of TMEM151B in zebrafish are still being elucidated, research on related transmembrane proteins suggests several potential roles:

Developmental Functions:
Transmembrane proteins in zebrafish often serve crucial roles in:

• Cell-cell communication during embryogenesis

• Tissue patterning and morphogenesis

• Neural development and circuit formation

• Regulation of developmental signaling pathways

Disease Modeling Applications:
Zebrafish provide excellent models for studying human disease relevance:

• Cancer Research:
  Zebrafish develop tumors in various tissues that are histologically similar to their mammalian counterparts. The zebrafish model has been used to identify cancer genes, including many that were previously unrecognized in mammals . Specific transmembrane proteins may function as:

  • Tumor suppressors

  • Oncogenes

  • Regulators of cell proliferation, migration, and invasion

• Neurological Disorders:
  Transmembrane proteins like TMEM119 show immunoreactivity in specific subsets of microglia in brains affected by neurodegenerative diseases such as Alzheimer's disease . TMEM151B may play similar roles in:

  • Neuronal development

  • Synaptic function

  • Glial cell activity

• Stress Response Pathways:
  Related proteins like Tmem39b protect zebrafish cells against DNA damage caused by environmental stressors and facilitate tissue damage repair . TMEM151B might function in:

  • Cellular stress responses

  • Tissue homeostasis maintenance

  • DNA damage repair mechanisms

To investigate these potential roles, researchers can employ:

• Loss-of-function studies using CRISPR-Cas9 or morpholinos

• Gain-of-function approaches through overexpression

• Small molecule screens to identify modulators of TMEM151B function

• Behavioral assays to assess neurological functions

What protein interaction studies can reveal TMEM151B function in zebrafish?

Several approaches can be employed to study TMEM151B protein interactions:

In Vivo Approaches:

• FRET (Fluorescence Resonance Energy Transfer):

  • Generate transgenic fish expressing TMEM151B fused to a donor fluorophore

  • Express potential interaction partners fused to acceptor fluorophores

  • Monitor energy transfer between fluorophores when proteins interact

  • This approach allows for real-time, in vivo monitoring of protein interactions in living zebrafish neurons

• Proximity Labeling:

  • Fuse TMEM151B to enzymes like BioID or APEX2

  • These enzymes biotinylate nearby proteins when activated

  • Identify labeled proteins by mass spectrometry

  • Provides a map of the TMEM151B protein interaction network

In Vitro Approaches:

• Co-Immunoprecipitation:

  • Extract proteins from zebrafish tissues

  • Use anti-TMEM151B antibodies to pull down the protein complex

  • Identify interacting partners by mass spectrometry

  • Validate interactions through reciprocal co-immunoprecipitation

• Yeast Two-Hybrid Screening:

  • Use TMEM151B domains as bait

  • Screen against zebrafish cDNA libraries

  • Validate positive interactions through secondary assays

• Protein Arrays:

  • Use purified recombinant TMEM151B to probe zebrafish protein arrays

  • Identify binding partners through direct protein-protein interactions

Functional Validation:

• Perform genetic interaction studies by combining tmem151b knockdown with manipulation of potential interacting partners

• Use small molecule inhibitors to disrupt specific interactions

• Employ domain mutation analysis to identify critical interaction motifs

These approaches can provide insights into the molecular networks and pathways involving TMEM151B in zebrafish development and disease.

How can high-throughput approaches be applied to study TMEM151B function in zebrafish?

Modern high-throughput approaches offer powerful ways to investigate TMEM151B:

Genomic Approaches:

• Genetic Screens:

  • Forward genetic screens using chemical mutagens or insertional mutagenesis

  • Reverse genetic screens using CRISPR-Cas9 libraries

  • Screens can be designed to identify modifiers of tmem151b phenotypes

  • Successful identification of mutations requires:

    • Rigorous phenotypic characterization

    • Calculation of logarithm of the odds (LOD) scores (≥3 considered proof of linkage)

    • Confirmation through complementation tests and rescue experiments

• RNA-Seq Analysis:

  • Compare transcriptomes between wild-type and tmem151b mutant zebrafish

  • Identify differentially expressed genes (DEGs) under various conditions

  • Perform pathway enrichment analysis to uncover affected biological processes

Proteomic Approaches:

• Quantitative Proteomics:

  • Use mass spectrometry-based approaches to profile protein changes

  • Compare proteomes between wild-type and tmem151b mutant fish

  • Correlate protein changes with transcriptional changes to identify post-transcriptional regulation

• Phosphoproteomics:

  • Analyze changes in protein phosphorylation patterns

  • Identify signaling pathways affected by TMEM151B manipulation

Functional Approaches:

• High-Content Imaging:

  • Automated microscopy to analyze cellular phenotypes

  • Live imaging of transgenic reporters in tmem151b backgrounds

  • Quantitative image analysis to detect subtle phenotypic changes

• Behavioral Screening:

  • Automated tracking of zebrafish locomotion and behaviors

  • Assess neurological functions in tmem151b mutants

  • High-throughput drug screening for compounds that modify phenotypes

Integration of Multi-Omics Data:

• Combine genomic, transcriptomic, and proteomic datasets

• Apply computational approaches to construct gene regulatory networks

• Identify key nodes and potential therapeutic targets

These high-throughput approaches can accelerate discovery of TMEM151B functions and provide comprehensive insights into its biological roles in zebrafish.