Recombinant Xenopus tropicalis Baculoviral IAP repeat-containing protein 7 (birc7)

What is birc7 and what are its key identifiers in Xenopus tropicalis?

Baculoviral IAP repeat containing 7 (birc7) is a protein-coding gene in Xenopus tropicalis that belongs to the Inhibitor of Apoptosis Protein (IAP) family. It is also known by several synonyms including EIAP/XLX, XLX, birc7-a, birc7-b, kiap, livin, ml-iap, mliap, rnf50, and xEIAP . The gene is assigned Entrez Gene ID 100127811 in genomic databases. As a member of the IAP family, birc7 is characterized by containing baculoviral IAP repeat domains that are critical for protein-protein interactions and its anti-apoptotic function.

The basic gene and protein identifiers for Xenopus tropicalis birc7 are summarized in the following table:

How does birc7 function in regulating apoptosis in Xenopus tropicalis?

Birc7 functions primarily as an inhibitor of apoptosis in Xenopus tropicalis by interfering with the caspase activation pathway. Like other IAP family members, birc7 likely inhibits both initiator and effector caspases through direct binding interactions. The anti-apoptotic function of IAPs is counterbalanced by pro-apoptotic proteins such as Diablo (also known as Smac).

In the Xenopus apoptotic pathway, cytochrome c release from mitochondria is a key event that triggers caspase activation. When cytochrome c is released, it forms a complex called the apoptosome, which activates caspases. IAP proteins, including birc7, act as a buffer preventing caspase activity and set an apoptotic threshold . This threshold appears to be critical in early development, as studies have shown that as little as 10 minutes of caspase activity is sufficient to cause apoptotic death in Xenopus oocytes .

The interplay between IAPs and their antagonists helps explain why there appears to be a concentration threshold for cytochrome c to induce apoptosis, with this threshold falling between 50 and 100 nM in Xenopus oocytes . This threshold can be lowered by Smac/Diablo, which binds to and neutralizes IAP proteins, thereby enhancing sensitivity to apoptotic stimuli.

What expression systems are most effective for producing recombinant Xenopus tropicalis birc7?

For recombinant expression of Xenopus tropicalis birc7, several expression systems can be employed with varying advantages depending on the experimental goals:

• Bacterial Expression System (E. coli):

  • Most commonly used for producing large quantities of protein for structural studies

  • Typically involves cloning the birc7 coding sequence into vectors like pET or pGEX

  • Expression often requires optimization of induction conditions (IPTG concentration, temperature, time)

  • Challenge: IAP proteins like birc7 may form inclusion bodies requiring refolding protocols

• Baculovirus Expression System:

  • Preferred for expressing eukaryotic proteins with proper folding and post-translational modifications

  • The birc7 cDNA ORF clone can be subcloned into baculovirus transfer vectors

  • Expression in insect cells (Sf9 or Hi5) yields proteins that more closely resemble native structure

  • Commercial cDNA ORF clones for Xenopus tropicalis birc7 are available for this purpose

• Mammalian Expression System:

  • Optimal for functional studies where proper folding and modification are critical

  • Common vectors include pcDNA and pCMV derivatives

  • Transient transfection in HEK293T or stable expression in CHO cells can be employed

  • Provides natural post-translational modifications but with lower yield than other systems

For most applications studying birc7 function, the baculovirus expression system offers an optimal balance between protein yield and proper folding. When initiating recombinant birc7 expression, verifying expression through Western blotting using either tag-specific antibodies or birc7-specific antibodies is essential for quality control.

How can I design loss-of-function experiments for birc7 in Xenopus tropicalis?

Designing effective loss-of-function experiments for birc7 in Xenopus tropicalis requires careful consideration of the approach and appropriate controls. Several methodological options are available:

• Morpholino Oligonucleotides:

  • Design translation-blocking or splice-blocking morpholinos targeting birc7 mRNA

  • Inject morpholinos into 1-2 cell stage embryos (typically 5-20 ng)

  • Include control morpholinos with similar chemical properties but non-targeting sequence

  • Validate knockdown efficiency through Western blotting or RT-PCR

  • Advantage: Rapid implementation and titrable dosage

• CRISPR/Cas9 Genome Editing:

  • Design guide RNAs targeting early exons of birc7

  • Co-inject Cas9 protein and guide RNAs into fertilized eggs

  • Screen F0 embryos for phenotypes and confirm editing by sequencing

  • Establish stable mutant lines through outcrossing and subsequent inbreeding

  • Advantage: Complete gene knockout possible compared to knockdown methods

  • Xenopus tropicalis is particularly amenable to genetic manipulation due to its diploid genome

• Dominant Negative Constructs:

  • Express truncated or mutated versions of birc7 lacking functional domains

  • These compete with endogenous birc7 for binding interactions

  • Microinject mRNA encoding dominant-negative constructs into embryos

  • Advantage: Can target specific protein-protein interactions

• Small Molecule Inhibitors:

  • Several IAP antagonists (e.g., SMAC mimetics) can be used

  • Apply at specific developmental stages to assess temporal requirements

  • Advantage: Allows precise temporal control of inhibition

For all approaches, validating specificity is crucial. This can be achieved through rescue experiments where wild-type birc7 mRNA (made resistant to the knockdown method) is co-injected to demonstrate phenotype reversal. Additionally, the gynogenetic screening method, which produces haploid embryos through UV-irradiated sperm fertilization followed by diploidization, can facilitate rapid identification of phenotypes without requiring extensive breeding .

What are the best methods for detecting apoptosis when studying birc7 function?

When studying birc7 function in regulating apoptosis in Xenopus tropicalis, multiple complementary approaches should be employed to detect and quantify apoptotic events:

• Live Imaging of Caspase Activity:

  • Microinjection of near-infrared fluorescent caspase substrates into oocytes or embryos

  • These substrates emit fluorescence only after proteolytic cleavage by active caspases

  • Allows real-time monitoring of caspase activation in living specimens

  • This approach has revealed that as little as 10 minutes of caspase activity can trigger irreversible apoptosis in Xenopus oocytes

• TUNEL Assay (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling):

  • Detects DNA fragmentation, a hallmark of apoptosis

  • Fix embryos or tissue sections and process with the TUNEL reaction mixture

  • Visualize labeled apoptotic cells through fluorescence microscopy

  • Quantify the percentage of TUNEL-positive cells in specific tissues

• Cleaved Caspase-3 Immunostaining:

  • Use antibodies specific for the activated (cleaved) form of caspase-3

  • Apply to fixed embryos or tissue sections

  • Analyze by fluorescence microscopy or flow cytometry

  • Provides direct evidence of caspase activation

• Annexin V Binding Assay:

  • Detects phosphatidylserine exposure on the outer leaflet of apoptotic cell membranes

  • Apply fluorescently labeled Annexin V to living cells or tissue

  • Combine with propidium iodide to distinguish early apoptotic from necrotic cells

  • Analyze by fluorescence microscopy or flow cytometry

• Mitochondrial Membrane Potential Assessment:

  • Use voltage-sensitive dyes like JC-1 or TMRE

  • Loss of mitochondrial membrane potential precedes cytochrome c release

  • Particularly relevant when studying IAP function in the intrinsic apoptotic pathway

When investigating birc7 specifically, combining these methods with protein interaction studies (co-immunoprecipitation with caspases or other IAP proteins) provides a comprehensive view of how birc7 regulates the apoptotic cascade in normal development or experimental conditions.

How does birc7 interact with other components of the apoptotic machinery?

Birc7 engages in a complex network of protein interactions within the apoptotic machinery of Xenopus tropicalis. According to protein interaction data, birc7 primarily functions through the following interactions:

• Interaction with Caspases:

  • Directly binds to and inhibits both initiator and effector caspases

  • The BIR (Baculoviral IAP Repeat) domains are essential for this interaction

  • Prevents proteolytic cleavage and activation of caspase cascades

• Antagonism by Diablo/Smac:

  • Diablo (also known as Smac) is a critical antagonist of IAP proteins including birc7

  • Diablo is released from mitochondria during apoptotic signaling

  • It binds to birc7 and other IAPs, preventing their interaction with caspases

  • This interaction has a very high confidence score (0.995) in protein interaction databases

  • The Diablo-IAP interaction helps establish the apoptotic threshold in Xenopus oocytes

• Interactions with Other IAP Family Members:

  • XIAP (xiap): Strong functional relationship with birc7, as both regulate caspase activity

  • Birc5l (Baculoviral IAP repeat-containing protein 5.2): Component of the chromosomal passenger complex with potential interaction with birc7

  • Birc2 and Birc6: Other IAP family members that may form heterotypic interactions

• Relationship with Cytochrome c Pathway:

  • Indirect interaction with cytochrome c signaling cascade

  • Cytochrome c (both cycs and cyct in Xenopus) triggers apoptosome formation and caspase activation

  • Birc7 acts downstream of cytochrome c release to regulate apoptotic outcomes

The functional significance of these interactions is evident in experiments showing that neutralizing IAPs with Smac/Diablo lowers the threshold concentration of cytochrome c needed to trigger apoptosis in Xenopus oocytes . This demonstrates the crucial buffering role that IAPs like birc7 play in setting apoptotic thresholds during development.

What is known about the differential functions of birc7-a and birc7-b isoforms?

Xenopus tropicalis expresses two isoforms of birc7: birc7-a and birc7-b . These isoforms likely arose through sub-functionalization following genome duplication events in the evolutionary history of Xenopus species. While detailed functional comparisons of these isoforms are still emerging, several key differences can be inferred:

• Expression Patterns:

  • The isoforms typically show divergent tissue-specific and developmental stage-specific expression patterns

  • This spatial and temporal regulation suggests specialized functions in different contexts

  • Quantitative PCR can be used to profile the relative abundance of each isoform across tissues and developmental stages

• Structural Distinctions:

  • While both contain the canonical BIR domains characteristic of IAP proteins, subtle differences in amino acid sequences may affect binding affinities to caspases and other interacting proteins

  • Differences in protein stability, post-translational modification sites, or subcellular localization signals may also exist between isoforms

• Functional Specialization:

  • Birc7-a may have retained more ancestral functions while birc7-b may have evolved specialized roles

  • This is consistent with the pattern observed in many duplicated genes in Xenopus tropicalis compared to the tetraploid X. laevis, where gene pairs often undergo subfunctionalization

• Compensation Mechanisms:

  • When one isoform is experimentally depleted, the other may show compensatory upregulation

  • This redundancy presents challenges for functional studies but can be addressed through simultaneous knockdown approaches

Experimental approaches to distinguish isoform-specific functions include:

• Generating isoform-specific antibodies for detection and localization studies

• Designing morpholinos or CRISPR guide RNAs that selectively target each isoform

• Performing rescue experiments with individual isoforms following double knockdown

• Using chimeric constructs to identify which domains contribute to isoform-specific functions

Understanding the distinct roles of birc7-a and birc7-b is particularly important given the value of Xenopus tropicalis as a model for studying gene evolution following duplication events.

How can high-throughput screening approaches identify novel regulators of birc7 activity?

High-throughput screening approaches offer powerful methods for identifying novel regulators of birc7 activity in Xenopus tropicalis. Several strategies can be implemented:

• Small Molecule Screening:

  • Establish a reporter system where birc7 activity influences a measurable output (e.g., caspase activity, cell viability)

  • Screen compound libraries in Xenopus tropicalis cell lines or in explanted tissues

  • Validate hits using dose-response studies and secondary assays

  • Advantage: May identify compounds with therapeutic potential for modulating apoptosis

• Genome-Wide CRISPR Screens:

  • Generate a library of guide RNAs targeting the Xenopus tropicalis genome

  • Introduce these into cells expressing a birc7 activity reporter

  • Use NGS to identify genes whose disruption alters birc7 function

  • This approach leverages the diploid nature of X. tropicalis, making it more amenable to genetic screens than the tetraploid X. laevis

• Protein Interaction Screening:

  • Use yeast two-hybrid or BioID approaches with birc7 as bait

  • Alternatively, perform immunoprecipitation coupled with mass spectrometry

  • Known interaction partners like Diablo/Smac and other IAP family members serve as positive controls

  • Novel interactions can be validated through co-immunoprecipitation and functional studies

• RNA-Seq After Perturbation:

  • Manipulate birc7 levels (overexpression or knockdown) at different developmental stages

  • Perform RNA-Seq to identify differentially expressed genes

  • Construct gene regulatory networks to identify feedback mechanisms

  • Use this approach to identify stage-specific dependencies on birc7 function

• Gynogenetic Screening for Genetic Modifiers:

  • Generate haploid embryos through UV-irradiated sperm fertilization

  • Diploidize using cold shock protocols to reveal recessive mutations

  • This method can rapidly identify genes that interact with birc7

  • The frequency of mutation appearance in gynogenetically-derived embryos depends on distance from the centromere, providing mapping information

These screening approaches are particularly powerful in Xenopus tropicalis due to its diploid genome, which facilitates genetic analysis, and the ease of generating large numbers of synchronously developing embryos for high-throughput studies.

How does birc7 function change during different developmental stages of Xenopus tropicalis?

The function of birc7 undergoes significant changes throughout Xenopus tropicalis development, reflecting the dynamic regulation of apoptosis required for proper embryogenesis. Key stage-specific functions include:

• Oocyte Stage:

  • IAP proteins including birc7 establish an apoptotic threshold in oocytes

  • This threshold requires a minimum concentration of cytochrome c (between 50-100 nM) to trigger apoptosis

  • IAPs act as a buffer preventing caspase activity below this threshold

• Meiotic Maturation:

  • Oocytes develop resistance to cytochrome c-induced apoptosis upon entry into meiosis

  • This represents a developmental checkpoint where apoptotic regulation shifts

  • The mechanism may involve post-translational modifications of birc7 or changes in its protein interactions

• Early Embryonic Development:

  • Apoptosis in very early development appears not to be cell-autonomous

  • The maternal-to-zygotic transition likely involves changes in birc7 regulation

  • Spatial patterns of birc7 expression become established as cell fates are specified

• Organogenesis:

  • Tissue-specific patterns of birc7 expression emerge

  • Apoptosis becomes critical for proper morphogenesis of organs

  • Birc7 may interact with tissue-specific factors to fine-tune apoptotic sensitivity

Research techniques to study these stage-specific changes include:

• Temporal RNA-Seq profiling to track changes in birc7 expression

• Stage-specific knockdown using photoactivatable morpholinos

• Protein-protein interaction studies at different developmental stages

• Live imaging of caspase activity using injected fluorescent substrates

Understanding the developmental regulation of birc7 is particularly valuable in Xenopus tropicalis because of its established role as a model for vertebrate development and the ability to directly visualize and manipulate embryos throughout development .

What are the implications of Xenopus tropicalis birc7 research for human disease models?

Research on Xenopus tropicalis birc7 has significant implications for understanding human diseases, particularly those involving dysregulation of apoptosis:

• Cancer Biology:

  • Human BIRC7 (Livin) is overexpressed in multiple cancer types

  • Mechanistic insights from Xenopus studies can inform therapeutic strategies

  • The high conservation of apoptotic pathways between Xenopus and humans makes findings translatable

  • Xenopus tropicalis offers advantages for screening potential therapeutic compounds targeting IAP proteins

• Developmental Disorders:

  • Disruptions in programmed cell death contribute to congenital abnormalities

  • Xenopus tropicalis allows visualization of developmental processes in real-time

  • The diploid genome of X. tropicalis facilitates genetic manipulation that more closely models human genetic conditions

  • Tissue chimeras can be readily created to study tissue-specific effects of birc7 mutations

• Degenerative Diseases:

  • Inappropriate activation of apoptosis contributes to neurodegenerative disorders

  • Understanding how birc7 regulates apoptotic thresholds may suggest neuroprotective strategies

  • The strong synteny between Xenopus tropicalis and mammalian genomes enhances the relevance of findings

• Regenerative Medicine:

  • Xenopus species exhibit remarkable regenerative capabilities

  • Birc7 may play roles in regulating cell survival during regeneration

  • Insights could inform approaches to enhance regeneration in human tissues

  • The ability to perform limb regeneration studies in X. tropicalis is particularly relevant

• Drug Discovery Pipeline:

  • Xenopus tropicalis embryos provide an excellent in vivo system for testing compounds targeting apoptotic pathways

  • High-throughput screening can identify small molecules that modulate birc7 function

  • Lead compounds can be rapidly assessed for developmental toxicity

  • The evolutionary conservation of IAP proteins increases the likelihood that findings will translate to human biology

The value of X. tropicalis as a model system stems from its combination of experimental tractability, diploid genome with strong synteny to mammals, and the ability to perform both genetic and embryological manipulations . These features make it an ideal bridge between simpler model organisms and mammalian systems for studying the fundamental mechanisms of apoptosis regulation by birc7 and other IAP proteins.

What are the main technical challenges in studying birc7 function and how can they be overcome?

Investigating birc7 function in Xenopus tropicalis presents several technical challenges, each requiring specific methodological solutions:

• Potential Redundancy Between IAP Family Members:

  • Challenge: Functional redundancy between birc7 and other IAPs (such as XIAP) may mask phenotypes in single-gene knockdown experiments

  • Solution: Implement combinatorial knockdown approaches targeting multiple IAPs simultaneously

  • Solution: Use domain-specific inhibitors that can target shared functional domains across IAP proteins

  • Solution: Perform careful dose-response studies to identify threshold effects in partial knockdowns

• Distinguishing Isoform-Specific Functions:

  • Challenge: The existence of birc7-a and birc7-b isoforms complicates functional analysis

  • Solution: Design isoform-specific morpholinos or CRISPR guide RNAs

  • Solution: Create isoform-specific antibodies for differential detection

  • Solution: Perform rescue experiments with individual isoforms to determine functional equivalence

• Temporal Regulation of Apoptosis:

  • Challenge: Birc7 function may vary across developmental stages

  • Solution: Use inducible or photoactivatable knockdown technologies

  • Solution: Employ stage-specific promoters to drive transgene expression

  • Solution: Implement the live imaging caspase activity assay to capture dynamic changes

• Spatial Heterogeneity:

  • Challenge: Tissue-specific effects may be missed in whole-embryo analyses

  • Solution: Implement tissue-specific CRISPR or transgene approaches

  • Solution: Use explant cultures to study tissue-specific responses

  • Solution: Apply single-cell RNA-seq to capture cellular heterogeneity

• Protein Stability and Turnover:

  • Challenge: IAP proteins may have complex regulation at the post-translational level

  • Solution: Use proteasome inhibitors to assess turnover rates

  • Solution: Create fusion proteins with destabilization domains for temporal control

  • Solution: Implement proximity labeling approaches to capture dynamic interaction partners

The technical advantages of Xenopus tropicalis as a model system help address many of these challenges. For example, the large embryo size facilitates microinjection of multiple reagents simultaneously, the external development allows continuous observation, and the diploid genome simplifies genetic approaches compared to X. laevis . Additionally, tissue explant and transplantation techniques provide powerful tools for dissecting tissue-specific functions of birc7.

How can contradictory results in birc7 functional studies be reconciled?

Contradictory results in birc7 functional studies can arise from multiple sources and require systematic approaches to reconcile:

• Developmental Stage Specificity:

  • Contradiction: Different phenotypes observed when manipulating birc7 at different stages

  • Resolution: Perform detailed time-course analyses with precisely timed interventions

  • Resolution: Use stage-specific markers to precisely define developmental context

  • Resolution: Implement inducible systems for temporal control of gene expression or inhibition

• Dosage Effects:

  • Contradiction: Varying degrees of knockdown or overexpression yielding different outcomes

  • Resolution: Conduct careful dose-response studies

  • Resolution: Use quantitative Western blotting to correlate protein levels with phenotypic outcomes

  • Resolution: Combine partial knockdowns with sensitized genetic backgrounds

• Maternal versus Zygotic Contributions:

  • Contradiction: Different results from morpholinos (affecting maternal and zygotic mRNAs) versus genetic mutations (primarily affecting zygotic function)

  • Resolution: Use methods that specifically target maternal transcripts

  • Resolution: Implement maternal-effect genetic screens using gynogenetic approaches

  • Resolution: Perform rescue experiments with mRNAs injected at different timepoints

• Isoform-Specific Functions:

  • Contradiction: Different outcomes when targeting birc7-a versus birc7-b

  • Resolution: Always specify which isoform is being studied

  • Resolution: Determine relative expression levels of each isoform in the tissue of interest

  • Resolution: Perform rescue experiments with each isoform individually

• Genetic Background Effects:

  • Contradiction: Different results in different laboratory strains

  • Resolution: Use inbred Xenopus tropicalis lines for consistent genetic background

  • Resolution: Test phenotypes in multiple genetic backgrounds

  • Resolution: Identify modifier loci through mapping approaches

• Compensatory Mechanisms:

  • Contradiction: Acute versus chronic loss of function yielding different phenotypes

  • Resolution: Compare morpholino knockdown (acute) with genetic mutants (chronic)

  • Resolution: Analyze transcriptional responses to identify compensatory gene expression

  • Resolution: Use rapid protein degradation approaches (e.g., auxin-inducible degron system)

The diploid nature of Xenopus tropicalis makes it particularly suitable for resolving such contradictions through genetic approaches, as mutations can be more readily mapped and characterized than in the tetraploid X. laevis . Additionally, the ability to generate tissue chimeras allows researchers to distinguish cell-autonomous from non-autonomous effects, which can be a source of apparent contradictions.

How can new genome editing technologies advance birc7 research in Xenopus tropicalis?

Advanced genome editing technologies offer unprecedented opportunities to study birc7 function in Xenopus tropicalis:

• Precision CRISPR/Cas9 Applications:

  • Base editors for introducing specific point mutations without DNA breaks

  • Prime editors for precise insertions or deletions without donor templates

  • These technologies allow creation of specific birc7 variants that mimic human disease mutations

  • Advantage: The diploid genome of X. tropicalis makes it more amenable to precise editing than X. laevis

• Inducible CRISPR Systems:

  • Light-activated or chemically-inducible Cas9 expression

  • Allows temporal control of birc7 disruption at specific developmental stages

  • Can be combined with tissue-specific promoters for spatiotemporal control

  • Helps resolve contradictory findings from constitutive knockout approaches

• CRISPR Interference/Activation (CRISPRi/CRISPRa):

  • Modulate birc7 expression levels without altering the genomic sequence

  • dCas9 fused to repressors (CRISPRi) or activators (CRISPRa) targeted to birc7 promoter regions

  • Enables study of dosage-dependent effects without complete loss of function

  • Can be applied to investigate isoform-specific regulation by targeting specific promoters

• Lineage Tracing with CRISPR:

  • CRISPR-based lineage recording systems (e.g., MEMOIR, LINNAEUS)

  • Track the fate of cells with birc7 mutations throughout development

  • Identify cell populations most sensitive to birc7 perturbation

  • Particularly powerful in Xenopus due to well-characterized cell lineages

• High-Throughput Mutagenesis:

  • Multiplex CRISPR screening targeting birc7 regulatory elements

  • Saturating mutagenesis of birc7 coding sequence to generate allelic series

  • Combined with gynogenetic screening for rapid phenotype identification

  • Enhances ability to identify critical functional domains and regulatory elements

These technologies are particularly valuable in Xenopus tropicalis due to its established position as a genetic model system with a sequenced genome, genetic mapping resources, and established transgenic methodologies . The combination of these genomic tools with the traditional strengths of Xenopus embryology creates powerful approaches for dissecting birc7 function in development and disease models.

What interdisciplinary approaches can provide new insights into birc7 biology?

Interdisciplinary approaches offer novel perspectives on birc7 biology in Xenopus tropicalis:

• Systems Biology Integration:

  • Multi-omics integration (transcriptomics, proteomics, metabolomics)

  • Network modeling of apoptotic regulatory circuits involving birc7

  • Identification of emergent properties not evident from single-gene studies

  • In silico prediction of condition-specific birc7 functions

• Structural Biology and Computational Modeling:

  • Cryo-EM or X-ray crystallography of Xenopus birc7 protein complexes

  • Molecular dynamics simulations of birc7 interactions with caspases and antagonists

  • Structure-based drug design targeting specific binding interfaces

  • Comparative structural analysis between Xenopus and human IAP proteins

• Synthetic Biology Approaches:

  • Engineer synthetic apoptotic circuits with defined components

  • Create optogenetic tools for spatial and temporal control of birc7 activity

  • Design synthetic IAP proteins with novel regulatory properties

  • Develop biosensors for real-time monitoring of birc7-protein interactions

• Evolutionary Developmental Biology:

  • Comparative analysis of birc7 function across amphibian species

  • Investigation of birc7 roles in regenerative versus non-regenerative contexts

  • Examination of birc7 subfunctionalization following genome duplication

  • The diploid genome of X. tropicalis makes it ideal for evolutionary comparisons with tetraploid X. laevis

• Quantitative Imaging and Biophysics:

  • Live imaging of fluorescent birc7 fusion proteins

  • Quantitative analysis of birc7 dynamics during apoptotic events

  • FRET-based sensors to detect birc7-caspase interactions in vivo

  • Super-resolution microscopy of birc7 subcellular localization

• Organoid and Ex Vivo Systems:

  • Development of Xenopus organoid cultures for tissue-specific studies

  • Ex vivo explant cultures to study birc7 function in specific tissues

  • Microfluidic systems for precise control of the cellular microenvironment

  • Combined with the live imaging caspase activity assay for real-time apoptosis monitoring

These interdisciplinary approaches leverage the unique advantages of Xenopus tropicalis as a model system, including its external development, accessible embryology, diploid genome, and evolutionary position . The integration of these approaches can provide a comprehensive understanding of birc7 biology that spans from molecular mechanisms to evolutionary significance.