Recombinant Xenopus tropicalis UPF0197 transmembrane protein C11orf10 homolog

Basic Research Questions

• What is Xenopus tropicalis UPF0197 transmembrane protein C11orf10 homolog and why is it studied?

The UPF0197 transmembrane protein C11orf10 homolog (also known as TMEM258) is a small transmembrane protein found in Xenopus tropicalis, the Western clawed frog. This protein is homologous to human TMEM258 (previously known as C11orf10). It consists of 79 amino acids with the sequence "MELEAMSRYTSPVNPAVFPHLTVVLLAIGMFFTAWFFVYEVTSTKYTRDVYKELLISLVASLFMGFGVLFLLLWVGIYV" .

Xenopus tropicalis serves as an excellent model organism for studying gene function due to its diploid genome that shows high conservation with human genes, making it valuable for understanding human genetic disorders . Researchers study this protein to understand its evolutionary conservation, membrane dynamics, and potential role in developmental processes.

• How is the gene for this protein identified and characterized in Xenopus tropicalis genome?

The tmem258 gene (Entrez Gene ID: 448722) in Xenopus tropicalis is also known by its synonym c11orf10 . Gene identification involves:

• Genome annotation through bioinformatics analysis

• Pfam domain identification to classify protein families

• Reciprocal BLAST searches between human and Xenopus tropicalis genomes

• Synteny analysis (examining conserved gene order) between mammalian and Xenopus genomes

Researchers often use the comprehensive Xenopus database (Xenbase) that provides accurate, annotated reference genome information with tools for genetic analysis and interpretation . The identification of transmembrane proteins typically involves hydrophobicity analysis to identify membrane-spanning domains.

• What expression patterns does tmem258 show during Xenopus tropicalis development?

While the specific expression pattern of tmem258 is not detailed in the provided search results, researchers typically analyze expression patterns through:

• In situ hybridization with RNA probes targeting tmem258

• RT-PCR analysis across developmental stages

• RNA-seq data analysis across embryonic timepoints

• Consultation of expression profiles available in Xenbase

Understanding expression patterns helps determine when and where the protein functions during development. To generate your own expression data, design antisense RNA probes that target tmem258 mRNA and perform whole-mount in situ hybridization on embryos at different developmental stages, from early cleavage through tadpole stages.

• What approaches can be used to produce recombinant Xenopus tropicalis TMEM258 protein?

Recombinant TMEM258 protein can be produced through several expression systems:

According to the search results, commercially available recombinant Xenopus tropicalis TMEM258 protein is typically produced with a purity of ≥85% as determined by SDS-PAGE . For optimal purification, researchers should include appropriate affinity tags (His, GST, or FLAG) and use detergents suitable for membrane proteins.

Advanced Research Questions

• How can CRISPR/Cas9 be used to investigate the function of tmem258 in Xenopus tropicalis?

CRISPR/Cas9 provides an efficient method for investigating tmem258 function:

Methodological approach:

• sgRNA design: Design sgRNAs targeting exons of tmem258 using online algorithms that predict cutting efficiencies

• Validation: Test multiple non-overlapping target sequences to ensure specificity

• Efficiency testing: Empirically determine cutting efficiency through Sanger sequencing of injected embryos followed by sequence-trace deconvolution

• Microinjection: Inject sgRNA with Cas9 protein into 1-cell or 2-cell stage embryos

• Unilateral mutation: Inject into one cell at the 2-cell stage to create embryos with one half carrying the mutation while the other half serves as an internal control

• Phenotyping: Analyze resulting phenotypes at appropriate developmental stages

This approach is particularly powerful in Xenopus tropicalis as thousands of mutant embryos can be generated in a single day, enabling parallel analysis of multiple genes .

• What are the best methods for determining membrane topology of Xenopus tropicalis TMEM258?

Determining membrane topology requires multiple complementary approaches:

• Computational prediction:

  • Use transmembrane prediction algorithms (TMHMM, Phobius)

  • Hydropathy analysis to identify hydrophobic transmembrane segments

• Experimental verification:

  • Protease protection assays: Treat intact vesicles with proteases to determine which regions are accessible

  • Glycosylation mapping: Add glycosylation sites at different positions to determine luminal exposure

  • Cysteine accessibility methods: Introduce cysteines and test their accessibility to membrane-impermeable reagents

  • Epitope tagging: Add epitope tags to N- and C-termini or internal loops and assess accessibility with antibodies

• Structural studies:

  • Cryo-electron microscopy if sufficient protein can be purified

  • NMR studies of reconstituted protein in membrane mimetics

For TMEM258, based on its small size (79 amino acids) , it likely contains 1-2 transmembrane domains, making it amenable to these approaches.

• What phenotypes result from knocking out tmem258 in Xenopus tropicalis?

While specific phenotypes for tmem258 knockout are not described in the search results, researchers can systematically analyze potential phenotypes:

• Embryonic development assessment:

  • Monitor morphological changes during development

  • Examine tissue-specific defects based on expression pattern

  • Analyze embryonic lethality or viability

• Molecular phenotyping:

  • Perform RNA-seq on knockout vs. control embryos

  • Use in situ hybridization to examine changes in marker gene expression

  • Conduct proteomic analysis to identify altered protein expression

• Functional assays:

  • Test membrane integrity and function in affected tissues

  • Examine cellular behaviors (migration, proliferation, apoptosis)

  • Evaluate response to environmental stressors

Xenopus tropicalis is particularly valuable for phenotypic analysis as embryos develop externally and rapidly, with organ systems forming within 4 days and quantifiable behaviors appearing within 10 days .

• How conserved is TMEM258 structure and function across vertebrate species?

Conservation analysis reveals important functional insights:

Structural conservation:

• The amino acid sequence from Xenopus tropicalis (79 amino acids) shows significant conservation with human TMEM258

• The gene is present across vertebrates, including mammals, amphibians, and fish species

• Conserved synteny (gene order) is observed between Xenopus and mammalian genomes

Functional conservation assessment methods:

• Multiple sequence alignment of TMEM258 proteins across species

• Phylogenetic analysis to determine evolutionary relationships

• Identification of conserved domains and critical residues

• Cross-species rescue experiments: Can human TMEM258 rescue Xenopus tmem258 knockout phenotypes?

Conservation suggests fundamental biological roles. The presence of TMEM258 homologs in species from zebrafish to humans indicates it likely serves important cellular functions preserved throughout vertebrate evolution.

• What protein-protein interactions have been identified for TMEM258 and how can they be studied in Xenopus?

To identify and study TMEM258 protein interactions:

Identification methods:

• Co-immunoprecipitation (Co-IP): Using tagged recombinant TMEM258 protein

• Proximity labeling: BioID or APEX2 fused to TMEM258 to identify proximal proteins

• Yeast two-hybrid screening: Using the soluble domains of TMEM258

• Mass spectrometry: After crosslinking and pull-down of TMEM258 complexes

Verification approaches:

• Fluorescence resonance energy transfer (FRET): To confirm direct interactions

• Bimolecular fluorescence complementation (BiFC): In Xenopus embryos

• Co-localization studies: Using fluorescently tagged proteins in Xenopus cells

Benefits of Xenopus system:

• Cell-free extract systems from Xenopus eggs provide a powerful environment for studying protein interactions

• Ability to express tagged proteins in developing embryos to observe interactions in vivo

• Rapid generation of mutants to assess the functional relevance of interactions

• How can recombinant Xenopus tropicalis TMEM258 be used in structural studies?

Structural determination of membrane proteins presents unique challenges:

Preparation strategies:

• Protein expression optimization:

  • Test multiple expression systems (E. coli, yeast, insect cells)

  • Use fusion partners to enhance solubility and folding

  • Include stabilizing mutations if necessary

• Purification approaches:

  • Solubilize with appropriate detergents (DDM, LMNG, etc.)

  • Use lipid nanodiscs or amphipols for native-like environment

  • Implement size exclusion chromatography for final purity

• Structural method selection:

  • X-ray crystallography (requires crystallization)

  • Cryo-electron microscopy (for larger complexes)

  • NMR spectroscopy (suitable for smaller membrane proteins like TMEM258)

  • Computational modeling validated by experimental data

The small size of TMEM258 (79 amino acids) makes it potentially amenable to NMR studies, especially if expressed with isotope labeling. Structural insights would significantly advance understanding of this protein's function.

• What experimental designs can determine the role of TMEM258 in early Xenopus development?

A comprehensive approach might include:

Loss-of-function studies:

• CRISPR/Cas9 knockout as described previously

• Morpholino antisense oligonucleotides targeting tmem258 mRNA

• Dominant negative constructs if appropriate binding partners are known

Gain-of-function approaches:

• mRNA overexpression at specific developmental stages

• Tissue-specific overexpression using appropriate promoters

• Expression of constitutively active forms if regulatory mechanisms are known

Rescue experiments:

• Co-injection of wild-type mRNA with CRISPR components

• Expression of orthologs from other species to test functional conservation

• Structure-function analysis with mutated forms of the protein

Downstream analysis:

• Time-lapse imaging of early development

• Cell lineage tracing in mutant vs. control embryos

• Transcriptomic analysis of affected tissues

• Biochemical assays for suspected molecular functions

Xenopus tropicalis is ideal for these studies as its embryos develop externally and can be readily manipulated and observed .

• How can evolutionary conservation analysis of tmem258 inform functional studies?

Evolutionary conservation analysis provides critical functional insights:

Analytical approaches:

• Sequence-based methods:

  • Multiple sequence alignment across diverse species

  • Identification of invariant amino acid residues

  • Calculation of conservation scores for each position

  • Prediction of functionally important regions

• Genomic context analysis:

  • Synteny conservation (neighboring genes)

  • Regulatory element conservation

  • Intron-exon structure comparison

Application to functional studies:

• Design mutations targeting highly conserved residues

• Prioritize protein domains with greatest conservation

• Predict functional motifs based on conserved sequences

• Guide the design of cross-species rescue experiments

Implementation in Xenopus:
Xenopus tropicalis serves as an excellent model for evolutionary studies due to its position in vertebrate phylogeny. The diploid genome of X. tropicalis shows high conservation with human genes, making it valuable for understanding human genetic disorders . This conservation allows researchers to translate findings between species and identify fundamental biological processes.

Technical and Methodological Questions

• What are the optimal conditions for expressing and purifying recombinant Xenopus tropicalis TMEM258?

Membrane protein expression and purification require specialized conditions:

Expression optimization:

Purification strategy:

• Cell lysis using methods gentle for membrane proteins

• Membrane fraction isolation through ultracentrifugation

• Solubilization with appropriate detergents (DDM, LMNG, etc.)

• Affinity chromatography using added tags (His, GST, etc.)

• Size exclusion chromatography for final purity

Quality control:

• SDS-PAGE analysis (should show ≥85% purity)

• Western blot confirmation of identity

• Mass spectrometry verification

• Functional assays if applicable

• What methods can be used to study TMEM258 localization in Xenopus tropicalis cells and tissues?

Multiple complementary approaches provide comprehensive localization data:

Imaging approaches:

• Immunofluorescence microscopy:

  • Using validated antibodies against TMEM258

  • Co-staining with organelle markers (ER, Golgi, plasma membrane)

  • Super-resolution techniques for detailed localization

• Live imaging with fluorescent fusion proteins:

  • GFP-tagged TMEM258 expression in embryos/cells

  • Time-lapse imaging to track dynamic localization

  • FRAP (Fluorescence Recovery After Photobleaching) for mobility assessment

• Electron microscopy:

  • Immunogold labeling for precise subcellular localization

  • Correlative light and electron microscopy (CLEM)

Biochemical approaches:

• Subcellular fractionation followed by Western blotting

• Surface biotinylation to determine plasma membrane localization

• Protease protection assays to determine topology

For Xenopus studies, whole-mount imaging of embryos allows visualization of expression patterns in developing tissues, while subcellular localization can be determined in cultured Xenopus cells or tissue sections.

• How can genome editing be used to introduce specific mutations or tags into the endogenous tmem258 gene?

Precise genome editing in Xenopus tropicalis enables sophisticated functional studies:

CRISPR/Cas9-mediated approaches:

• Point mutations introduction:

  • Design sgRNA targeting desired location

  • Provide single-stranded oligodeoxynucleotide (ssODN) repair template with mutation

  • Screen for precise edits using restriction fragment length polymorphism (RFLP) or sequencing

• Epitope or fluorescent protein tagging:

  • Design sgRNAs near terminus to be tagged

  • Provide repair template with tag sequence and homology arms

  • Verify incorporation by PCR, Western blot, or fluorescence

• Conditional allele creation:

  • Insert loxP sites flanking critical exons

  • Express Cre recombinase tissue-specifically

  • Verify tissue-specific deletion

Practical considerations for Xenopus:

• F0 embryos often show mosaicism; breeding to establish stable lines is recommended

• Gynogenesis protocols can expedite generation of homozygous mutants by skipping a generation

• Multiple sgRNAs can be used to increase editing efficiency

• Microinjection into 1-cell stage embryos ensures widespread distribution of editing components

The ability to rapidly generate and screen large numbers of embryos makes Xenopus tropicalis an excellent system for optimization of genome editing strategies.

Frequently Asked Questions about Recombinant Xenopus tropicalis UPF0197 Transmembrane Protein C11orf10 Homolog

Basic Research Questions

• What is Xenopus tropicalis UPF0197 transmembrane protein C11orf10 homolog and why is it studied?

The UPF0197 transmembrane protein C11orf10 homolog (also known as TMEM258) is a small transmembrane protein found in Xenopus tropicalis, the Western clawed frog. This protein is homologous to human TMEM258 (previously known as C11orf10). It consists of 79 amino acids with the sequence "MELEAMSRYTSPVNPAVFPHLTVVLLAIGMFFTAWFFVYEVTSTKYTRDVYKELLISLVASLFMGFGVLFLLLWVGIYV" .

Xenopus tropicalis serves as an excellent model organism for studying gene function due to its diploid genome that shows high conservation with human genes, making it valuable for understanding human genetic disorders . Researchers study this protein to understand its evolutionary conservation, membrane dynamics, and potential role in developmental processes.

• How is the gene for this protein identified and characterized in Xenopus tropicalis genome?

The tmem258 gene (Entrez Gene ID: 448722) in Xenopus tropicalis is also known by its synonym c11orf10 . Gene identification involves:

• Genome annotation through bioinformatics analysis

• Pfam domain identification to classify protein families

• Reciprocal BLAST searches between human and Xenopus tropicalis genomes

• Synteny analysis (examining conserved gene order) between mammalian and Xenopus genomes

Researchers often use the comprehensive Xenopus database (Xenbase) that provides accurate, annotated reference genome information with tools for genetic analysis and interpretation . The identification of transmembrane proteins typically involves hydrophobicity analysis to identify membrane-spanning domains.

• What expression patterns does tmem258 show during Xenopus tropicalis development?

While the specific expression pattern of tmem258 is not detailed in the provided search results, researchers typically analyze expression patterns through:

• In situ hybridization with RNA probes targeting tmem258

• RT-PCR analysis across developmental stages

• RNA-seq data analysis across embryonic timepoints

• Consultation of expression profiles available in Xenbase

Understanding expression patterns helps determine when and where the protein functions during development. To generate your own expression data, design antisense RNA probes that target tmem258 mRNA and perform whole-mount in situ hybridization on embryos at different developmental stages, from early cleavage through tadpole stages.

• What approaches can be used to produce recombinant Xenopus tropicalis TMEM258 protein?

Recombinant TMEM258 protein can be produced through several expression systems:

According to the search results, commercially available recombinant Xenopus tropicalis TMEM258 protein is typically produced with a purity of ≥85% as determined by SDS-PAGE . For optimal purification, researchers should include appropriate affinity tags (His, GST, or FLAG) and use detergents suitable for membrane proteins.

Advanced Research Questions

• How can CRISPR/Cas9 be used to investigate the function of tmem258 in Xenopus tropicalis?

CRISPR/Cas9 provides an efficient method for investigating tmem258 function:

Methodological approach:

• sgRNA design: Design sgRNAs targeting exons of tmem258 using online algorithms that predict cutting efficiencies

• Validation: Test multiple non-overlapping target sequences to ensure specificity

• Efficiency testing: Empirically determine cutting efficiency through Sanger sequencing of injected embryos followed by sequence-trace deconvolution

• Microinjection: Inject sgRNA with Cas9 protein into 1-cell or 2-cell stage embryos

• Unilateral mutation: Inject into one cell at the 2-cell stage to create embryos with one half carrying the mutation while the other half serves as an internal control

• Phenotyping: Analyze resulting phenotypes at appropriate developmental stages

This approach is particularly powerful in Xenopus tropicalis as thousands of mutant embryos can be generated in a single day, enabling parallel analysis of multiple genes .

• What are the best methods for determining membrane topology of Xenopus tropicalis TMEM258?

Determining membrane topology requires multiple complementary approaches:

• Computational prediction:

  • Use transmembrane prediction algorithms (TMHMM, Phobius)

  • Hydropathy analysis to identify hydrophobic transmembrane segments

• Experimental verification:

  • Protease protection assays: Treat intact vesicles with proteases to determine which regions are accessible

  • Glycosylation mapping: Add glycosylation sites at different positions to determine luminal exposure

  • Cysteine accessibility methods: Introduce cysteines and test their accessibility to membrane-impermeable reagents

  • Epitope tagging: Add epitope tags to N- and C-termini or internal loops and assess accessibility with antibodies

• Structural studies:

  • Cryo-electron microscopy if sufficient protein can be purified

  • NMR studies of reconstituted protein in membrane mimetics

For TMEM258, based on its small size (79 amino acids) , it likely contains 1-2 transmembrane domains, making it amenable to these approaches.

• What phenotypes result from knocking out tmem258 in Xenopus tropicalis?

While specific phenotypes for tmem258 knockout are not described in the search results, researchers can systematically analyze potential phenotypes:

• Embryonic development assessment:

  • Monitor morphological changes during development

  • Examine tissue-specific defects based on expression pattern

  • Analyze embryonic lethality or viability

• Molecular phenotyping:

  • Perform RNA-seq on knockout vs. control embryos

  • Use in situ hybridization to examine changes in marker gene expression

  • Conduct proteomic analysis to identify altered protein expression

• Functional assays:

  • Test membrane integrity and function in affected tissues

  • Examine cellular behaviors (migration, proliferation, apoptosis)

  • Evaluate response to environmental stressors

Xenopus tropicalis is particularly valuable for phenotypic analysis as embryos develop externally and rapidly, with organ systems forming within 4 days and quantifiable behaviors appearing within 10 days .

• How conserved is TMEM258 structure and function across vertebrate species?

Conservation analysis reveals important functional insights:

Structural conservation:

• The amino acid sequence from Xenopus tropicalis (79 amino acids) shows significant conservation with human TMEM258

• The gene is present across vertebrates, including mammals, amphibians, and fish species

• Conserved synteny (gene order) is observed between Xenopus and mammalian genomes

Functional conservation assessment methods:

• Multiple sequence alignment of TMEM258 proteins across species

• Phylogenetic analysis to determine evolutionary relationships

• Identification of conserved domains and critical residues

• Cross-species rescue experiments: Can human TMEM258 rescue Xenopus tmem258 knockout phenotypes?

Conservation suggests fundamental biological roles. The presence of TMEM258 homologs in species from zebrafish to humans indicates it likely serves important cellular functions preserved throughout vertebrate evolution.

• What protein-protein interactions have been identified for TMEM258 and how can they be studied in Xenopus?

To identify and study TMEM258 protein interactions:

Identification methods:

• Co-immunoprecipitation (Co-IP): Using tagged recombinant TMEM258 protein

• Proximity labeling: BioID or APEX2 fused to TMEM258 to identify proximal proteins

• Yeast two-hybrid screening: Using the soluble domains of TMEM258

• Mass spectrometry: After crosslinking and pull-down of TMEM258 complexes

Verification approaches:

• Fluorescence resonance energy transfer (FRET): To confirm direct interactions

• Bimolecular fluorescence complementation (BiFC): In Xenopus embryos

• Co-localization studies: Using fluorescently tagged proteins in Xenopus cells

Benefits of Xenopus system:

• Cell-free extract systems from Xenopus eggs provide a powerful environment for studying protein interactions

• Ability to express tagged proteins in developing embryos to observe interactions in vivo

• Rapid generation of mutants to assess the functional relevance of interactions

• How can recombinant Xenopus tropicalis TMEM258 be used in structural studies?

Structural determination of membrane proteins presents unique challenges:

Preparation strategies:

• Protein expression optimization:

  • Test multiple expression systems (E. coli, yeast, insect cells)

  • Use fusion partners to enhance solubility and folding

  • Include stabilizing mutations if necessary

• Purification approaches:

  • Solubilize with appropriate detergents (DDM, LMNG, etc.)

  • Use lipid nanodiscs or amphipols for native-like environment

  • Implement size exclusion chromatography for final purity

• Structural method selection:

  • X-ray crystallography (requires crystallization)

  • Cryo-electron microscopy (for larger complexes)

  • NMR spectroscopy (suitable for smaller membrane proteins like TMEM258)

  • Computational modeling validated by experimental data

The small size of TMEM258 (79 amino acids) makes it potentially amenable to NMR studies, especially if expressed with isotope labeling. Structural insights would significantly advance understanding of this protein's function.

• What experimental designs can determine the role of TMEM258 in early Xenopus development?

A comprehensive approach might include:

Loss-of-function studies:

• CRISPR/Cas9 knockout as described previously

• Morpholino antisense oligonucleotides targeting tmem258 mRNA

• Dominant negative constructs if appropriate binding partners are known

Gain-of-function approaches:

• mRNA overexpression at specific developmental stages

• Tissue-specific overexpression using appropriate promoters

• Expression of constitutively active forms if regulatory mechanisms are known

Rescue experiments:

• Co-injection of wild-type mRNA with CRISPR components

• Expression of orthologs from other species to test functional conservation

• Structure-function analysis with mutated forms of the protein

Downstream analysis:

• Time-lapse imaging of early development

• Cell lineage tracing in mutant vs. control embryos

• Transcriptomic analysis of affected tissues

• Biochemical assays for suspected molecular functions

Xenopus tropicalis is ideal for these studies as its embryos develop externally and can be readily manipulated and observed .

• How can evolutionary conservation analysis of tmem258 inform functional studies?

Evolutionary conservation analysis provides critical functional insights:

Analytical approaches:

• Sequence-based methods:

  • Multiple sequence alignment across diverse species

  • Identification of invariant amino acid residues

  • Calculation of conservation scores for each position

  • Prediction of functionally important regions

• Genomic context analysis:

  • Synteny conservation (neighboring genes)

  • Regulatory element conservation

  • Intron-exon structure comparison

Application to functional studies:

• Design mutations targeting highly conserved residues

• Prioritize protein domains with greatest conservation

• Predict functional motifs based on conserved sequences

• Guide the design of cross-species rescue experiments

Implementation in Xenopus:
Xenopus tropicalis serves as an excellent model for evolutionary studies due to its position in vertebrate phylogeny. The diploid genome of X. tropicalis shows high conservation with human genes, making it valuable for understanding human genetic disorders . This conservation allows researchers to translate findings between species and identify fundamental biological processes.

Technical and Methodological Questions

• What are the optimal conditions for expressing and purifying recombinant Xenopus tropicalis TMEM258?

Membrane protein expression and purification require specialized conditions:

Expression optimization:

Purification strategy:

• Cell lysis using methods gentle for membrane proteins

• Membrane fraction isolation through ultracentrifugation

• Solubilization with appropriate detergents (DDM, LMNG, etc.)

• Affinity chromatography using added tags (His, GST, etc.)

• Size exclusion chromatography for final purity

Quality control:

• SDS-PAGE analysis (should show ≥85% purity)

• Western blot confirmation of identity

• Mass spectrometry verification

• Functional assays if applicable

• What methods can be used to study TMEM258 localization in Xenopus tropicalis cells and tissues?

Multiple complementary approaches provide comprehensive localization data:

Imaging approaches:

• Immunofluorescence microscopy:

  • Using validated antibodies against TMEM258

  • Co-staining with organelle markers (ER, Golgi, plasma membrane)

  • Super-resolution techniques for detailed localization

• Live imaging with fluorescent fusion proteins:

  • GFP-tagged TMEM258 expression in embryos/cells

  • Time-lapse imaging to track dynamic localization

  • FRAP (Fluorescence Recovery After Photobleaching) for mobility assessment

• Electron microscopy:

  • Immunogold labeling for precise subcellular localization

  • Correlative light and electron microscopy (CLEM)

Biochemical approaches:

• Subcellular fractionation followed by Western blotting

• Surface biotinylation to determine plasma membrane localization

• Protease protection assays to determine topology

For Xenopus studies, whole-mount imaging of embryos allows visualization of expression patterns in developing tissues, while subcellular localization can be determined in cultured Xenopus cells or tissue sections.

• How can genome editing be used to introduce specific mutations or tags into the endogenous tmem258 gene?

Precise genome editing in Xenopus tropicalis enables sophisticated functional studies:

CRISPR/Cas9-mediated approaches:

• Point mutations introduction:

  • Design sgRNA targeting desired location

  • Provide single-stranded oligodeoxynucleotide (ssODN) repair template with mutation

  • Screen for precise edits using restriction fragment length polymorphism (RFLP) or sequencing

• Epitope or fluorescent protein tagging:

  • Design sgRNAs near terminus to be tagged

  • Provide repair template with tag sequence and homology arms

  • Verify incorporation by PCR, Western blot, or fluorescence

• Conditional allele creation:

  • Insert loxP sites flanking critical exons

  • Express Cre recombinase tissue-specifically

  • Verify tissue-specific deletion

Practical considerations for Xenopus:

• F0 embryos often show mosaicism; breeding to establish stable lines is recommended

• Gynogenesis protocols can expedite generation of homozygous mutants by skipping a generation

• Multiple sgRNAs can be used to increase editing efficiency

• Microinjection into 1-cell stage embryos ensures widespread distribution of editing components