Recombinant Mouse Granulocyte colony-stimulating factor protein (Csf3) (Active)

How does recombinant mouse CSF3 compare functionally to human CSF3?

While both human and mouse CSF3 share functional homology in stimulating granulocyte production, several key differences affect their experimental applications:

Mouse CSF3 acts primarily on mouse cells, though with some cross-reactivity to human cells. Notably, human G-CSF can be used in mouse models such as BDF1 mice, which show a stimulatory response to human G-CSF . This cross-species activity is particularly valuable when evaluating novel delivery methods or fusion proteins.

For experimental design, it's important to note that human G-CSF fusion proteins (like G-CSF-Tf) administered to mice at 5 mg/kg (0.05 μmol/kg) subcutaneously or 50 mg/kg (0.5 μmol/kg) orally have demonstrated effective myelopoietic activity . These dosing parameters can serve as starting points when designing mouse experiments with either human or mouse CSF3 variants.

What are the validated methods for measuring recombinant mouse CSF3 bioactivity?

The standard method for assessing CSF3 bioactivity is the NFS-60 cell proliferation assay. This methodology involves:

• Washing NFS-60 cells three times with RPMI medium 1640/10% FBS

• Plating cells in 96-well microtiter plates at 1 × 10^5 cells per ml

• Adding 10-fold serial dilutions of CSF3 protein

• Incubating at 37°C in 5% CO2 for 48 hours

• Performing an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay:

  • Treating cells with 1 mg/ml MTT in serum-free, phenol red-free medium for 4 hours

  • Dissolving formazan crystals in isopropanol

  • Measuring absorbance at 570 nm

For more advanced applications, researchers can evaluate:

• STAT3/STAT5 nuclear translocation in transfected cells (e.g., HeLa cells expressing CSF3R)

• JAK activation via phosphorylation status

• Receptor internalization and recycling dynamics

• ROS production in myeloid cells (which involves the Lyn-PI3-kinase-Akt pathway)

How should storage and handling protocols be optimized for maintaining recombinant mouse CSF3 activity?

Recombinant mouse CSF3 requires careful handling as it is "an active protein and may elicit a biological response in vivo" . Optimal storage and handling protocols include:

• Store at -80°C for long-term storage

• Prepare single-use aliquots to avoid repeated freeze-thaw cycles

• Reconstitute in sterile buffer (PBS or manufacturer's recommended buffer)

• For dilute solutions, add carrier protein (e.g., 0.1% BSA) to prevent adsorption loss

• For quality control validation, recommended techniques include:

  • SDS-PAGE analysis

  • ESI-TOF mass spectrometry verification (predicted MW: 19008.20 Da)

  • HPLC analysis for purity assessment

What strategies have been developed for enhancing delivery and bioavailability of CSF3?

Recent advances in CSF3 delivery include fusion protein technology that significantly improves administration options:

The G-CSF-transferrin (G-CSF-Tf) fusion protein represents a breakthrough in oral delivery of CSF3. This construct is engineered by fusing human cDNAs encoding G-CSF and transferrin with a dipeptide linker (Leu-Glu) . Unlike chemical conjugation methods that produce heterogeneous mixtures, this recombinant approach yields a consistent product.

Expression in HEK293 cells using protein-free medium allows harvesting of the fusion protein after 5 days, with isolation via ammonium sulfate precipitation. When administered orally, G-CSF-Tf demonstrates sustained myelopoietic effects lasting up to 3 days, compared to the shorter duration of subcutaneously administered native G-CSF .

This research demonstrates that transferrin can function as an effective carrier for oral delivery of therapeutic proteins, opening possibilities for other fusion protein constructs with improved bioavailability .

How do mutations in CSF3R affect signaling and what experimental approaches best characterize these alterations?

CSF3R mutations have significant implications for signaling dynamics and are associated with several hematological disorders. Key mutations and their experimental characterization include:

• Truncation Mutations:

  • Nonsense mutations truncating the carboxyl-terminus of CSF3R occur in approximately 30% of severe congenital neutropenia (SCN) patients

  • These mutations increase to approximately 80% after progression to acute myeloid leukemia (AML)

  • Experimental approach: Compare wild-type and truncated CSF3R signaling in myeloid progenitor proliferation assays

• Activating Point Mutations:

  • T595I mutation: Found in both SCN/AML and de novo AML patients

  • T617I and T617N mutations: Located in the transmembrane domain

  • These mutations cause ligand-independent activation of CSF3R

• Experimental Characterization Approaches:

  • Site-directed mutagenesis to create specific mutations (e.g., CSF3R-T595V construct to test hydrophobicity effects)

  • Transfection studies in relevant cell lines

  • Colony formation assays to assess autonomous proliferation

  • Analysis of STAT3/STAT5 nuclear accumulation in the absence of CSF3 stimulation

In mutation studies, substituting threonine for valine at position 595 (T595V) resulted in growth factor-independent progenitor cell proliferation similar to T595I, indicating that hydrophobicity rather than structural changes drives autonomous signaling .

How does recombinant mouse CSF3 contribute to modeling hematological disorders?

Recombinant mouse CSF3 is instrumental in developing models for various hematological conditions:

• Severe Congenital Neutropenia (SCN) Models:

  • CSF3 is clinically used to treat patients with SCN (neutrophil counts <0.5 million/L)

  • Mouse models with ELANE or HAX1 mutations (common in SCN) can be treated with recombinant CSF3 to study neutrophil response patterns

  • These models help assess how long-term CSF3 therapy might contribute to leukemic progression

• Leukemia Progression Models:

  • The acquisition of CSF3R mutations represents a significant step in leukemic transformation

  • Studies comparing SCN patients in neutropenia phase versus after progression to AML reveal important insights into mutation accumulation

  • Research shows that CSF3R-T595I mutation was a late event, detected after 17 years of CSF3 therapy in one documented SCN/AML patient

• Reactive Oxygen Species (ROS) Mechanisms:

  • CSF3-induced ROS production involves the Lyn-PI3-kinase-Akt pathway

  • This mechanism may contribute to genotoxic damage and potential carcinogenesis

  • Mouse models can help elucidate how truncated CSF3R proteins might exacerbate ROS production and DNA damage

What considerations should guide experimental design when studying CSF3 signaling in neutrophil development?

When designing experiments to study CSF3's role in neutrophil development, researchers should consider:

• Signal Activation and Attenuation Balance:

  • CSF3 signaling requires "a tight but dynamic balance between signal activation and attenuation" to achieve appropriate neutrophil output

  • Experiments should assess both activation pathways and negative feedback mechanisms

  • Time-course studies are essential as signaling dynamics change over time

• Receptor Expression and Trafficking:

  • Monitor CSF3R expression levels and localization

  • Consider receptor internalization, degradation, and recycling

  • Account for potential expression of truncated or mutant receptors

• Species-Specific Considerations:

  • While mouse models provide valuable insights, species differences exist

  • For translational research, validation in human cells is recommended

  • BDF1 mice respond to human G-CSF and can serve as useful models

• Dosing Parameters:

  • Different administration routes require adjusted dosing

  • Subcutaneous: 1 mg/kg (G-CSF) or 5 mg/kg (G-CSF-Tf)

  • Oral: 10 mg/kg (G-CSF) or 50 mg/kg (G-CSF-Tf)

  • Duration of treatment influences outcomes (acute vs. chronic exposure)

• Readout Selection:

  • Cell proliferation assays (e.g., NFS-60 cells)

  • Colony formation in semi-solid media

  • Flow cytometry for neutrophil maturation stages

  • Signaling pathway activation (JAK-STAT, Lyn-PI3K-Akt)

  • ROS production measurement

What are the common challenges in recombinant mouse CSF3 experiments and how can they be addressed?

Researchers working with recombinant mouse CSF3 frequently encounter several challenges:

• Activity Loss During Storage:

  • Problem: Decreased bioactivity over time

  • Solution: Store at -80°C in single-use aliquots; add carrier protein for dilute solutions; verify activity via NFS-60 proliferation assay before critical experiments

• Variable Cellular Response:

  • Problem: Inconsistent proliferation or differentiation results

  • Solution: Characterize CSF3R expression levels in your cell model; ensure cells haven't developed CSF3 independence; verify batch consistency using control cell lines

• Species Cross-Reactivity Issues:

  • Problem: Unexpected differences when translating between mouse and human systems

  • Solution: Validate cross-species activity before experimental design; consider species-specific receptors and downstream signaling differences

• Mutation Detection Challenges:

  • Problem: Difficulty identifying CSF3R mutations

  • Solution: Use denaturing high-performance liquid chromatography (dHPLC) followed by Sanger sequencing of samples showing aberrant patterns

How can researchers verify the structural integrity and activity of recombinant mouse CSF3 preparations?

Quality control for recombinant mouse CSF3 requires multiple complementary approaches:

• Structural Verification:

  • Mass spectrometry (ESI-TOF) to confirm molecular weight (expected: 19008.20 ±10 Da)

  • SDS-PAGE analysis to assess purity (should be ≥95%)

  • Western blotting using specific antibodies against mouse CSF3

• Functional Validation:

  • NFS-60 cell proliferation assay (gold standard for bioactivity)

  • MTT assay following 48-hour incubation with serial dilutions

  • Comparison to a reference standard with known activity

• Purity Assessment:

  • HPLC analysis to confirm absence of contaminants

  • Endotoxin testing (should be ≤0.005 EU/μg)

  • Sterility testing for cell culture applications

• Post-Translational Modification Analysis:

  • Verification of O-glycosylation patterns

  • Assessment of proper disulfide bond formation

  • Confirmation of correct protein folding via circular dichroism or other structural techniques

By implementing these comprehensive quality control measures, researchers can ensure consistent and reliable results when working with recombinant mouse CSF3 protein in their experimental systems.