VEGF Mouse, Yeast

What is VEGF and what are its primary functions in mouse models?

VEGF (Vascular Endothelial Growth Factor) is a signaling protein family that stimulates vasculogenesis and angiogenesis. The VEGF protein family consists of six members: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and Placental Growth Factor (PGF) . VEGF-A is the most extensively studied member, particularly for its role in stimulating endothelial cell mitogenesis and cell migration.

In mouse models, VEGF proteins serve several critical physiological functions:

• Creating new blood vessels during embryonic development

• Facilitating formation of new vessels after tissue injury

• Developing new vessels in exercised muscle

• Establishing collateral circulation to bypass blocked vessels

VEGF-A was originally referred to as vascular permeability factor (VPF) due to its ability to increase microvascular permeability. While primarily studied for its effects on vascular endothelium, VEGF-A also influences monocyte/macrophage migration, neurons, cancer cells, and kidney epithelial cells .

How do transgenic VEGF-GFP mouse models work and what information do they provide?

Transgenic VEGF-GFP mouse models contain a construct where green fluorescent protein (GFP) expression is driven by the VEGF promoter, allowing for in situ monitoring of VEGF gene transcription. These models provide a visual reporter system that illuminates when and where VEGF is being actively transcribed in living tissues.

In successful VEGF-GFP models, GFP fluorescence patterns closely match tissues known to express VEGF mRNA, including:

• Lung, cartilage, and brain in neonatal mice

• Moderate fluorescence in the upper epidermis

• Prominent expression in outer root sheath keratinocytes of hair follicles

• Strong up-regulation in hyperplastic epidermis at wound edges

These models allow researchers to:

• Observe real-time VEGF transcription in living mice

• Distinguish between transcriptional regulation and mRNA stability effects

• Monitor VEGF expression changes during development, injury, and experimental treatments

• Localize VEGF transcription to specific cell populations

Why are yeast expression systems preferred for producing recombinant VEGF proteins?

Yeast expression systems, particularly Pichia pastoris, offer significant advantages over bacterial systems like E. coli for recombinant VEGF production:

The recombinant mouse VEGF-A produced in Pichia pastoris has a predicted molecular weight of 19.3 kDa and can be used for various research applications including cell culture, ELISA standards, and Western blot controls .

How are VEGF-GFP transgenic mice generated and what promoter regions are most effective?

Creating VEGF-GFP transgenic mice involves several critical steps requiring careful molecular design:

• Promoter selection: A 2.453-kb fragment (-2,362 to +91) of the 5′-upstream region of the human VEGF promoter has proven effective .

• Vector construction: This involves:

  • PCR amplification of the VEGF promoter region

  • Insertion of the promoter fragment into a GFP vector (such as pEGFP-1)

  • Creating a transgene construct (designated as pVEGF-GFP)

• Transgenic mouse generation:

  • Excision of the transgene fragment from the vector

  • Microinjection into fertilized oocytes (often DBA2×C57BL/6 mice)

  • Implantation into pseudopregnant foster mothers

  • Identification of founder mice (F0) via genomic PCR and Southern blot analysis

The effectiveness of the promoter can be validated through in vitro transfection experiments prior to mouse generation. Notably, the VEGF promoter shows differential activity depending on cell type, with stronger expression in keratinocytes compared to dermal fibroblasts .

What are the most effective methods for monitoring VEGF expression in transgenic mouse models?

Multiple complementary approaches allow for comprehensive monitoring of VEGF expression in transgenic models:

VEGF-GFP expression can be detected as early as 6 hours after topical TPA treatment in the epidermis of mouse ear skin using confocal laser microscopy. This allows for early detection of changes in VEGF gene transcription in response to stimuli .

What experimental controls are essential when working with VEGF-GFP mouse models?

Robust experimental design with appropriate controls is crucial for meaningful results with VEGF-GFP mice:

• Genetic controls:

  • Wild-type littermates to establish background autofluorescence levels

  • Mice with different transgene copy numbers to assess dose-dependent effects

• Experimental validation controls:

  • In situ hybridization for VEGF mRNA to confirm correlation with GFP fluorescence

  • Comparison of GFP signal with known VEGF expression patterns in tissues like lung, cartilage, and brain

• Treatment controls:

  • Vehicle-only treatments (e.g., acetone for TPA experiments)

  • Time-matched untreated controls

  • Dose-response assessments for stimuli

• Technical controls:

  • Consistent imaging parameters across experimental groups

  • Inclusion of fluorescence standards for quantitative comparisons

  • Multiple tissue sections and biological replicates

When examining wound-induced VEGF expression, researchers observed strong up-regulation of GFP fluorescence in hyperplastic epidermis at the wound edge 48 hours after wounding, with minimal fluorescence in the dermis. This pattern was confirmed by in situ hybridization for VEGF mRNA, validating the model's accuracy .

What specific advantages does Pichia pastoris offer for recombinant VEGF production compared to other expression systems?

Pichia pastoris offers distinct advantages for VEGF production that make it particularly suitable for research applications:

• Proper protein folding: As a eukaryotic organism, P. pastoris possesses cellular machinery that facilitates correct folding of complex proteins like VEGF, with appropriate disulfide bond formation.

• Post-translational modifications: P. pastoris can perform essential glycosylation that affects VEGF bioactivity, unlike bacterial systems.

• High purity preparation: Recombinant proteins from P. pastoris are endotoxin-free and can be purified without HIS-TAGS or other tags that might interfere with protein function .

• Secretion capacity: P. pastoris efficiently secretes recombinant proteins into the culture medium, simplifying downstream purification.

• Bioactivity preservation: The yeast expression system produces VEGF proteins that more closely resemble their host form in terms of structure and function .

These advantages make P. pastoris-produced VEGF particularly valuable for sensitive applications such as cell-based assays, where endotoxin contamination or structural alterations could confound results.

What protocols yield the highest quality recombinant mouse VEGF-A from yeast systems?

Optimized protocols for high-quality mouse VEGF-A production in yeast systems include:

• Expression optimization:

  • Selection of appropriate P. pastoris strain

  • Codon optimization of the VEGF-A gene for yeast expression

  • Use of strong, inducible promoters

  • Cultivation at lower temperatures (20-25°C) during induction phase

• Purification strategy:

  • Harvesting the secreted protein from culture supernatant

  • Initial clarification through centrifugation and filtration

  • Chromatographic purification without requiring affinity tags

  • Final polishing steps to achieve high purity

• Quality control measures:

  • Verification of the predicted molecular weight (19.3 kDa for mouse VEGF-A)

  • Confirmation of proper folding and biological activity

  • Endotoxin testing to ensure preparation is endotoxin-free

  • Stability assessment under various storage conditions

The reconstitution of lyophilized VEGF-A should be performed in sterile PBS with at least 0.1% carrier protein (such as BSA) or in cell assay media to maintain stability and activity .

How can researchers validate the biological activity of yeast-produced VEGF proteins?

Validation of yeast-produced VEGF biological activity requires multiple complementary approaches:

• In vitro endothelial cell assays:

  • Proliferation assays measuring BrdU incorporation or MTT conversion

  • Migration assays using Boyden chambers or wound healing models

  • Tube formation assays on Matrigel to assess angiogenic potential

• Receptor activation assessment:

  • Western blot analysis of VEGFR2 phosphorylation

  • Examination of downstream signaling pathway activation (Erk1/2, PLCγ)

  • Dose-response relationships for receptor activation

• Comparative analysis:

  • Side-by-side comparison with mammalian cell-produced VEGF

  • Benchmarking against commercial standards

  • Cross-validation in multiple assay systems

• In vivo validation:

  • Mouse ear vasculature assay using adenoviral vectors expressing VEGF-A164

  • Corneal pocket angiogenesis assays

  • Matrigel plug assays for neovascularization

Activity assessment should be quantitative, with appropriate statistical analysis to ensure the recombinant protein meets or exceeds established activity standards.

How can VEGF-GFP transgenic mice be used to study pathological angiogenesis?

VEGF-GFP transgenic mice provide powerful tools for investigating pathological angiogenesis in various disease models:

• Cancer research applications:

  • Real-time monitoring of VEGF expression during tumor development

  • Evaluation of anti-angiogenic therapies on VEGF transcription

  • Identification of cell populations responsible for VEGF production in the tumor microenvironment

  • Correlation of VEGF expression patterns with tumor progression stages

• Inflammation and wound healing:

  • Visualization of VEGF upregulation in hyperplastic epidermis at wound edges

  • Tracking VEGF expression during different phases of the wound healing process

  • Assessment of how inflammatory stimuli like TPA induce VEGF expression

  • Study of potential anti-inflammatory interventions on VEGF-driven angiogenesis

• Ischemic conditions:

  • Monitoring VEGF expression in response to tissue hypoxia

  • Evaluation of therapeutic angiogenesis approaches

  • Investigation of collateral vessel formation mechanisms

The temporal resolution offered by these models allows researchers to determine precisely when VEGF expression changes occur in relation to pathological processes, providing insights into causality rather than mere correlation.

What are the key VEGF signaling pathways that can be studied using combined mouse models and yeast-produced proteins?

Research combining transgenic mouse models with yeast-produced VEGF proteins can elucidate several critical signaling pathways:

• VEGFR2 (KDR/Flk-1) signaling cascade:

  • PLCγ activation leading to calcium signaling and PKC activation

  • Erk1/2 phosphorylation promoting proliferation

  • PI3K/Akt pathway activation enhancing survival

  • p38 MAPK stimulation influencing migration

• Receptor cross-talk and modulation:

  • VEGFR1/VEGFR2 interactions

  • Neuropilin co-receptor influences

  • Receptor internalization and recycling dynamics

• Signaling inhibition and modulation:

  • Effects of probiotic yeast (S. boulardii) on VEGFR signaling

  • Pharmacological intervention points

  • Feedback mechanisms regulating receptor activity

The use of highly purified, yeast-produced VEGF proteins allows for precise stimulation of these pathways under controlled conditions, while transgenic reporters enable visualization of downstream effects in complex tissues.

How does probiotic yeast influence VEGF signaling and angiogenesis?

Recent research indicates that probiotic yeast, specifically S. boulardii, can modulate VEGF-mediated angiogenesis through several mechanisms:

• Regulation of VEGFR signaling:

  • Modulation of VEGFR phosphorylation

  • Influence on downstream signaling components including PLCγ and Erk1/2

  • Potential receptor internalization or degradation effects

• Physiological consequences:

  • Limitation of intestinal inflammation

  • Promotion of mucosal tissue repair

  • Potential normalization of pathological angiogenesis

• Experimental approaches to study this interaction:

  • Mouse ear vasculature assay with adenoviral VEGF-A164 expression

  • Western blot analysis of signaling components

  • Cell-based assays for angiogenic responses

This emerging area represents a novel intersection between probiotic research and angiogenesis biology, with potential therapeutic implications for inflammatory bowel diseases and other conditions characterized by pathological angiogenesis.

What are common challenges in generating consistent VEGF expression data from mouse models?

Researchers frequently encounter several challenges when working with VEGF-GFP mouse models:

The search results indicate that transgenic mice exhibited variable GFP-derived fluorescence across tissues, with stronger expression in certain cell types like outer root sheath keratinocytes compared to dermal fibroblasts. This cell-type specificity must be considered when interpreting results .

What quality control measures ensure reliable results with yeast-produced VEGF proteins?

To ensure reliable results with yeast-produced VEGF proteins, researchers should implement these quality control measures:

• Structural integrity assessment:

  • SDS-PAGE to confirm the predicted molecular weight (19.3 kDa for mouse VEGF-A)

  • Mass spectrometry to verify protein identity and detect modifications

  • Circular dichroism to assess secondary structure

• Functional validation:

  • Dose-dependent activation of VEGF receptors

  • Comparison with commercially available standards

  • Consistent results across multiple production batches

• Contaminant testing:

  • Endotoxin testing using LAL assay

  • Host cell protein quantification

  • Aggregation assessment through size exclusion chromatography

• Storage stability:

  • Activity testing after various storage conditions

  • Freeze-thaw stability assessment

  • Proper reconstitution protocols with carrier proteins like BSA

Careful quality control not only ensures experimental reproducibility but also prevents misleading results due to protein degradation or contamination.

How can researchers resolve contradictory data between VEGF expression in vitro versus in vivo models?

When faced with contradictions between in vitro and in vivo VEGF expression data, researchers should consider:

• Physiological context differences:

  • In vitro systems lack complex tissue microenvironments

  • Oxygen tension differences affect VEGF expression

  • Growth factor and cytokine networks present in vivo are absent in vitro

• Methodological approaches to resolve discrepancies:

  • Validate in vitro findings with ex vivo tissue explants as an intermediate step

  • Use conditional expression systems that function in both contexts

  • Compare multiple cell types in vitro to match the heterogeneity of in vivo tissues

  • Employ tissue-specific primary cells rather than immortalized lines

• Technical considerations:

  • Standardize detection methods across experimental systems

  • Use multiple complementary techniques (e.g., fluorescence, in situ hybridization, immunohistochemistry)

  • Account for differences in protein stability and turnover between systems

The search results demonstrate that the VEGF promoter activity was stronger in transfected keratinocytes than in dermal fibroblasts in vitro, which correlated with the pattern observed in vivo where epidermal keratinocytes showed stronger GFP expression than dermal cells . This alignment between in vitro and in vivo observations helps validate the model system.

What emerging technologies are enhancing VEGF mouse models?

Recent technological advances are creating new opportunities for VEGF research:

• CRISPR/Cas9 gene editing:

  • Creation of more precise knock-in reporter models

  • Generation of conditional VEGF expression systems

  • Introduction of specific VEGF isoforms or mutations

• Advanced imaging approaches:

  • Intravital microscopy for longitudinal studies

  • Light-sheet microscopy for 3D tissue analysis

  • Multicolor reporters for simultaneous visualization of multiple factors

• Single-cell technologies:

  • Single-cell RNA sequencing to identify heterogeneous VEGF-expressing populations

  • Spatial transcriptomics to map VEGF expression in tissue contexts

  • CyTOF and spectral flow cytometry for multiparameter analysis

These technologies will allow researchers to address more sophisticated questions about VEGF biology in development, disease, and therapeutic contexts.

How can combined mouse-yeast research systems advance therapeutic applications?

The integration of mouse models and yeast expression systems creates powerful platforms for therapeutic development:

• Drug screening and validation:

  • Use of yeast-produced VEGF to screen potential inhibitors in vitro

  • Validation of promising compounds in VEGF-GFP mouse models

  • Monitoring of both target engagement and downstream effects

• Biologics development:

  • Engineering of VEGF variants with modified properties in yeast

  • Testing engineered variants in mouse models for desired effects

  • Development of VEGF-targeted antibodies and other biologics

• Combination therapy approaches:

  • Investigation of probiotic yeast effects on VEGF signaling for inflammatory conditions

  • Exploration of synergistic approaches combining VEGF modulation with other pathways

  • Development of tissue-specific delivery strategies

This integrative approach accelerates translation from basic mechanistic insights to clinical applications by providing both versatile production systems and relevant in vivo models.

What are the most promising directions for understanding VEGF regulation through yeast-based systems?

Several promising research directions are emerging at the intersection of yeast biology and VEGF regulation:

• Probiotic mechanisms:

  • Further elucidation of how S. boulardii regulates VEGFR signaling

  • Identification of specific bioactive compounds produced by yeast

  • Investigation of yeast-host interactions affecting VEGF expression

• Metabolic influences:

  • Study of how yeast-derived metabolites affect VEGF signaling

  • Exploration of shared regulatory pathways between yeast and mammalian cells

  • Investigation of the microbiome-angiogenesis axis

• Production system innovations:

  • Development of yeast strains with humanized glycosylation patterns

  • Creation of inducible expression systems with precise control

  • Engineering of yeast to produce novel VEGF variants with modified properties

These directions represent fertile ground for interdisciplinary research that could yield both fundamental insights and practical applications in therapeutic angiogenesis and anti-angiogenic approaches.