Recombinant Mouse Fibroblast growth factor 2 (Fgf2) (Active)

What is recombinant mouse FGF2 and how does it differ from FGF2 from other species?

Recombinant mouse FGF2 (also known as basic FGF or bFGF) is one of 22 mitogenic proteins in the FGF family that show 35-60% amino acid conservation. The 17 kDa mouse FGF2 sequence has 98% amino acid identity with rat and 95% identity with human, bovine, and sheep FGF basic proteins. Unlike many other growth factors, FGF2 lacks a conventional signal peptide and is secreted through an alternative pathway, often utilizing cellular storage pools or binding to cell surface heparan sulfate proteoglycans (HSPGs) . This high degree of conservation suggests that mouse FGF2 may be substituted with human FGF2 in some experimental contexts, although species-specific differences should be considered for sensitive applications .

What are the structural characteristics of recombinant mouse FGF2?

Recombinant mouse FGF2 is typically produced as a protein spanning amino acids Met1-Ser154 or Ala11-Ser154, with a molecular weight of approximately 17.2 kDa, though it often appears at around 16-18 kDa on SDS-PAGE. The commercially available protein is commonly produced in E. coli expression systems, purified to ≥95% purity, and contains minimal endotoxin contamination (<1.0 EU/μg) . The protein requires binding to heparin or cell surface HSPGs for proper receptor binding, dimerization, and activation of tyrosine kinase FGF receptors. Additionally, FGF2 can bind to other proteins, polysaccharides, and lipids with lower affinity, which can influence its biological activity in different experimental contexts .

What is the biological activity range for recombinant mouse FGF2 in standard assays?

The biological activity of recombinant mouse FGF2 is typically measured using cell proliferation assays with mouse fibroblast cell lines such as BALB/c 3T3 or NR6R-3T3 cells. The effective dose that induces 50% of maximal response (ED50) typically ranges from 0.3-1.8 ng/mL for high-quality preparations . This activity measure serves as an important quality control parameter for researchers selecting a recombinant protein preparation. When designing dose-response experiments, researchers should consider testing a range spanning at least one order of magnitude above and below this ED50 value to fully capture the biological response curve in their specific experimental system .

How does FGF2 signaling affect neurogenesis and cognitive function in neurodegenerative disease models?

FGF2 plays a critical role in neurogenesis and cognitive function, particularly in neurodegenerative disease models. Studies using AAV2/1-mediated FGF2 gene delivery in APP+presenilin-1 (PS1) bigenic mice (an Alzheimer's disease model) have demonstrated significant improvements in spatial learning as measured by radial arm water maze tests. This improvement correlates with enhanced numbers of doublecortin, BrdU/NeuN, and c-fos–positive cells in the dentate gyrus, indicating increased neurogenesis and neuronal activation .

Specifically, the percentage of BrdU+/NeuN+ cells (representing newly generated neurons) in the dentate gyrus was approximately 16.8% in non-transgenic mice, decreased to 16.4% and 10.9% in AAV2/1-GFP–injected APP+PS1 mice (presymptomatic and postsymptomatic treatment, respectively), but increased to 20.1% and 13.8% after AAV2/1-FGF2 injections in presymptomatic and postsymptomatic treatments, respectively . These findings suggest that FGF2 can promote neuronal stem cell proliferation and differentiation in Alzheimer's disease models, potentially counteracting the neurodegeneration-associated reduction in neurogenesis.

What are the molecular mechanisms by which FGF2 enhances amyloid-β clearance in Alzheimer's disease models?

FGF2 enhances amyloid-β (Aβ) clearance through multiple complementary mechanisms. In APP+PS1 mice, AAV2/1-FGF2 gene delivery was shown to enhance the clearance of fibrillar Aβ in the hippocampus. In vitro studies have demonstrated that FGF2 enhances Aβ phagocytosis in primary cultured microglia, suggesting a direct effect on microglial function and clearance capacity . Additionally, FGF2 can reduce Aβ production from primary cultured neurons after AAV2/1-FGF2 infection, indicating that it may also modulate APP processing or Aβ generation pathways .

How does disruption of the FGF2 gene affect mouse development and physiology compared to other FGF family members?

Despite the near-ubiquitous expression of FGF2, disruption of the mouse FGF2 gene results in a relatively mild phenotype. This suggests significant functional compensation by other FGF family members . The mild phenotype observed in FGF2 knockout mice contrasts with the more severe developmental effects seen when FGF receptors are disrupted, further supporting the notion of redundancy among FGF ligands.

In contrast, transgenic overexpression of FGF2 results in excessive cellular proliferation and angiogenesis, reminiscent of various pathological conditions . This differential impact between gene deletion and overexpression highlights the importance of precise regulation of FGF2 signaling in normal physiology. Researchers investigating FGF2 function should consider potential compensatory mechanisms when interpreting knockout studies and may benefit from using combined knockouts of multiple FGF family members or conditional/inducible approaches to more fully elucidate FGF2's specific functions .

What are the optimal storage and reconstitution conditions for recombinant mouse FGF2?

Recombinant mouse FGF2 is typically provided in lyophilized form and requires proper reconstitution and storage for maintaining biological activity. The recommended reconstitution protocol varies slightly between preparations:

For carrier protein-containing preparations:

• Reconstitute at 100 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin .

• Store the reconstituted protein at -20°C to -80°C.

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles to maintain activity .

For carrier-free preparations:

• Reconstitute at 100 μg/mL in sterile PBS .

• Some formulations may be in specific buffers like 20mM PB, 400mM NaCl, pH 7.0 .

• Store the reconstituted protein at -20°C to -80°C.

• Aliquot before freezing to minimize freeze-thaw cycles .

Before use in cell culture, it's advisable to sterile filter through a 0.2 μm filter. For long-term storage beyond 1 month, storage at -80°C is recommended, while working aliquots may be kept at -20°C for up to 1 month .

How should recombinant mouse FGF2 be used in neuronal cell culture systems?

When using recombinant mouse FGF2 in neuronal cell culture systems, several methodological considerations are important:

• Concentration titration: While the general ED50 for FGF2 is 0.3-1.8 ng/mL in fibroblast proliferation assays, neuronal systems may require different concentrations. Research indicates that neuronal responses may require concentrations in the 5-50 ng/mL range .

• Heparin supplementation: Consider adding heparin (1-10 μg/mL) to culture media, as it can stabilize FGF2 and enhance its binding to FGF receptors, particularly in defined media lacking heparan sulfate proteoglycans .

• Treatment timing: For neuronal differentiation studies, the timing of FGF2 addition is critical. Some studies suggest that FGF2 may inhibit neuronal differentiation when applied during certain developmental windows but promote neurite outgrowth and survival at others .

• Co-factors: For neural stem cell experiments, consider combining FGF2 with EGF (10-20 ng/mL) as this combination often yields optimal neurosphere formation and neural stem cell expansion .

• Media considerations: Use low-protein or serum-free media for clearer interpretation of FGF2-specific effects, as serum contains various growth factors that may confound results .

What are the best methods for detecting the biological activity of recombinant mouse FGF2 in experimental systems?

Several robust methods exist for assessing recombinant mouse FGF2 biological activity:

• Cell proliferation assays: The standard bioactivity assay uses mouse fibroblast cell lines (NR6R-3T3, BALB/c 3T3) to measure proliferation in response to FGF2. A dose-response curve can be generated using concentrations from 0.1-10 ng/mL, with expected ED50 values of 0.3-1.8 ng/mL .

• Neurosphere formation assay: For neural applications, the ability of FGF2 to promote neurosphere formation from neural stem/progenitor cells provides a functional readout. Typically, 10-20 ng/mL FGF2 is used, often in combination with EGF .

• Phosphorylation of downstream signaling molecules: Western blotting for phosphorylated ERK1/2, AKT, or FRS2 at 5-30 minutes post-treatment provides a rapid biochemical assessment of FGF receptor activation. This approach is particularly useful when testing FGF2 activity in new experimental systems .

• Neurite outgrowth: In neuronal cultures, measuring neurite length, branching, or complexity after 24-72 hours of FGF2 treatment provides a functional readout relevant to neuronal development and regeneration .

• Doublecortin or BrdU/NeuN immunostaining: In vivo or in neuronal cultures, these markers can be used to assess neurogenesis in response to FGF2 treatment, with expected increases of approximately 20-30% in positive cells compared to controls .

Why might recombinant mouse FGF2 show reduced or variable activity in cell culture experiments?

Several factors can contribute to reduced or variable activity of recombinant mouse FGF2 in cell culture:

• Protein degradation: FGF2 is susceptible to degradation, particularly in serum-free conditions. Adding a carrier protein (0.1-1% BSA) to the culture medium can help stabilize the protein .

• Inadequate co-factors: FGF2 requires heparan sulfate proteoglycans (HSPGs) for optimal receptor binding and activation. In defined media lacking these components, adding heparin (1-10 μg/mL) can enhance FGF2 activity .

• Receptor downregulation: Prolonged exposure to FGF2 can lead to receptor downregulation and decreased responsiveness. Consider using pulsed treatment regimens rather than continuous exposure in long-term experiments .

• Cell density effects: Both too high and too low cell densities can affect responses to FGF2. Optimize seeding density for each cell type and assay system .

• Formulation differences: Carrier-free versus BSA-containing preparations may exhibit different activities in certain experimental systems. The BSA-containing preparation is generally recommended for cell culture applications, while carrier-free versions are preferable when BSA might interfere with the experimental system .

How can researchers distinguish between the effects of endogenous and exogenously added recombinant mouse FGF2?

Distinguishing between endogenous and exogenous FGF2 effects can be challenging but several approaches can help:

• Use of neutralizing antibodies: Specific antibodies against mouse FGF2 can be used to block endogenous FGF2 activity before adding tagged or human recombinant FGF2 that might be less affected by the antibody.

• Gene silencing approaches: siRNA or shRNA against endogenous FGF2 can reduce background levels before adding exogenous protein.

• CRISPR/Cas9 gene editing: Creating FGF2 knockout cell lines provides a clean background for testing exogenous FGF2.

• Tagged recombinant proteins: Using His-tagged or otherwise modified recombinant FGF2 can allow specific detection and potentially different activity profiles compared to endogenous protein.

• Receptor inhibitors: Using specific inhibitors of FGF receptors can establish baseline signaling before adding exogenous FGF2 at concentrations that potentially overcome the inhibition.

• Time-course analysis: Endogenous FGF2 often establishes a steady-state level of signaling, while exogenous addition produces acute signaling peaks that can be distinguished temporally .

What are potential cross-reactions or interference issues when studying recombinant mouse FGF2 in complex biological systems?

When studying recombinant mouse FGF2 in complex biological systems, several cross-reactions or interference issues should be considered:

• FGF receptor promiscuity: FGF2 can bind and activate multiple FGF receptors (FGFR1-4) with different affinities, potentially triggering diverse and sometimes conflicting cellular responses. This is particularly important in heterogeneous cell populations where different receptor profiles may exist .

• Interaction with other growth factors: FGF2 signaling pathways can cross-talk with other growth factor pathways (e.g., EGF, PDGF, VEGF), potentially synergizing or antagonizing their effects. Consider controlled experiments with single factors versus combinations .

• Matrix interactions: FGF2 binds to extracellular matrix components, particularly heparan sulfate proteoglycans, which can modulate its availability and activity. The composition of the extracellular environment can therefore significantly impact experimental outcomes .

• Endogenous FGF2 production: Many cell types produce endogenous FGF2, which can mask or complicate the interpretation of exogenous recombinant FGF2 effects. Background levels should be assessed and controlled for .

• Isoform-specific effects: Mouse FGF2 has multiple isoforms with different subcellular localizations and functions. Standard recombinant proteins typically represent the 17-18 kDa secreted form, but larger nuclear-localized isoforms (21-24 kDa) exist endogenously and may have distinct functions .

How is recombinant mouse FGF2 being utilized in neurodegenerative disease research?

Recombinant mouse FGF2 has emerged as a promising tool in neurodegenerative disease research, particularly for Alzheimer's disease (AD). Research has demonstrated several important applications:

• Gene therapy approaches: AAV2/1-mediated FGF2 gene delivery to the hippocampi of APP+PS1 bigenic mice (an AD model) has been shown to significantly improve spatial learning in the radial arm water maze test when administered at both pre- and post-symptomatic stages. This approach resulted in long-term expression of FGF2 (1,550-1,957 pg/mg at 4-24 weeks post-injection compared to 516.8-684.5 pg/mg in control injected animals) .

• Neural stem cell stimulation: FGF2 has been shown to enhance neurogenesis in the dentate gyrus, with AAV2/1-FGF2 injection increasing the number of doublecortin-positive cells (a marker of immature neurons) and BrdU/NeuN-positive cells (newly generated mature neurons) .

• Amyloid clearance: FGF2 promotes clearance of fibrillar amyloid-β peptide in the hippocampus, potentially through enhancing microglial phagocytosis and reducing Aβ production from neurons .

• Synaptic plasticity improvement: FGF2 gene delivery enhances long-term potentiation (LTP) in APP mouse models (J20), which correlates with increased expression of c-fos (a marker of neuronal activity) .

These findings suggest that recombinant FGF2 or FGF2 gene delivery could potentially serve as an alternative therapy for Alzheimer's disease and possibly other neurocognitive disorders .

What are the latest findings regarding FGF2's role in tissue regeneration and repair mechanisms?

Recent research has expanded our understanding of FGF2's critical role in tissue regeneration and repair:

• Bone regeneration: Single-nucleus transcriptomics has revealed that FGF2 influences differentiation trajectories of periosteal skeletal/stem progenitor cells during bone regeneration . FGF2 appears to promote proliferation of these progenitor populations while maintaining their multi-potentiality.

• Angiogenesis modulation: FGF2 has well-established pro-angiogenic effects that are being exploited in regenerative medicine approaches. Recent work has focused on controlled delivery systems that can provide sustained, localized FGF2 release to promote vascularization of injured or engineered tissues .

• Neural regeneration: Beyond its neurogenic effects, FGF2 has been shown to promote axonal regeneration and remyelination after injury. Research is exploring how FGF2 interacts with extracellular matrix components in the inhibitory environment of CNS injuries .

• Wound healing: FGF2 plays multiple roles in cutaneous wound healing, affecting keratinocytes, fibroblasts, and endothelial cells. Recent work has focused on how the timing and concentration of FGF2 application can shift the balance between regenerative healing versus fibrotic scarring .

• Interactions with inflammatory processes: FGF2's regenerative effects are now understood to be closely linked to its immunomodulatory properties, with emerging evidence that it can promote anti-inflammatory M2 macrophage polarization in some contexts .

How do different experimental designs affect the interpretation of recombinant mouse FGF2 function in developmental biology studies?

Experimental design significantly impacts the interpretation of recombinant mouse FGF2 function in developmental biology studies:

• Timing of FGF2 administration: FGF2's effects are highly context-dependent and can vary dramatically based on developmental stage. During early neural development, FGF2 may promote neural precursor proliferation, while at later stages it may inhibit terminal differentiation of these precursors . Carefully controlled timing of FGF2 administration is therefore critical for reproducible results.

• Concentration-dependent effects: FGF2 often exhibits bell-shaped dose-response curves, with different concentrations promoting distinct cellular behaviors. Low concentrations (0.1-1 ng/mL) may promote survival, moderate concentrations (1-10 ng/mL) may optimize proliferation, while higher concentrations (>50 ng/mL) may induce differentiation or other responses in certain cell types .

• Delivery methods: Different delivery methods (bolus addition, sustained release, genetic overexpression) can dramatically alter FGF2's effects. For example, AAV2/1-mediated FGF2 gene delivery produces sustained expression (1,812, 1,550, and 1,957 pg/mg at 4, 12, and 24 weeks post-injection, respectively) that may better mimic physiological signaling than bolus recombinant protein addition .

• Receptor expression patterns: The developmental expression patterns of FGF receptors (FGFR1-4) vary across tissues and developmental stages. Experimental interpretation must consider which receptors are expressed in the system under study and how they might mediate different responses to FGF2 .

• Interactions with extracellular matrix: The composition of the extracellular environment, particularly the presence of specific heparan sulfate proteoglycans, dramatically influences FGF2 activity. In vitro studies using defined media may not fully recapitulate the complex extracellular environment in which FGF2 naturally functions during development .