sRANKL Mouse
Description
Production and Recombinant Variants
sRANKL Mouse is commercially produced for research, with notable variants:
Recombinant sRANKL is non-glycosylated, lyophilized, and reconstituted in sterile buffers for experimental use .
Biological Functions
sRANKL Mouse mediates two primary pathways:
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Osteoclastogenesis:
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Immune Modulation:
Bone Loss and Osteoporosis Models
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sRANKL Overexpression: Transgenic mice (sRANKL-TG) develop severe osteoporosis due to excessive osteoclast activity .
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Hypercalcemia Induction: Intraperitoneal injection of adenovirus-expressed sRANKL (Ad-sRANKL) rapidly elevates serum calcium, mimicking human humoral hypercalcemia of malignancy (HHM) .
Osteopetrosis Models
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Anti-RANKL Antibodies: Single-dose administration (e.g., OYC1 monoclonal antibody) blocks sRANKL, increasing cancellous bone mass in adult mice .
Developmental Studies
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Age-Dependent Effects:
Pharmacological Inhibition Studies
Small-molecule inhibitors like S3-15 selectively target sRANKL:
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Binding Mechanism: Stabilizes sRANKL’s enlarged binding pocket, forming H-bonds with D303 and interacting with K180/Q236 .
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Potency: IC₅₀ = 0.096 μM (osteoclastogenesis inhibition), KD = 34.80 μM (binding affinity) .
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Pharmacokinetics: Oral availability confirmed in murine models .
Key Research Findings
Clinical Relevance
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Denosumab Analogues: While denosumab (human anti-RANKL antibody) doesn’t cross-react with murine sRANKL, chimeric mouse-human models (HuRANKL mice) enable preclinical testing .
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Therapeutic Targeting: sRANKL-specific inhibitors like S3-15 offer potential for treating osteoporosis with reduced off-target effects .
Product Specs
Recombinant mouse sRANKL, a non-glycosylated polypeptide chain consisting of 174 amino acids (143-316 a.a.), is produced in E. coli. With a molecular weight of 19.9 kDa, this protein variant undergoes purification using proprietary chromatographic techniques to isolate the CD254 molecule.
This protein solution (1mg/ml) is lyophilized in a buffer of 10mM Na2PO4 at pH 7.5 and 50mM NaCl.
To reconstitute the lyophilized sRANKL, it is recommended to dissolve it in sterile 18MΩ-cm H2O to a concentration of at least 100µg/ml. The resulting solution can be further diluted in other aqueous solutions as needed.
Lyophilized TNFSF11 can be stored at room temperature for up to 3 weeks, but for extended storage, it should be kept desiccated at a temperature below -18°C. After reconstitution, sRANKL should be stored at 4°C for no more than 2-7 days. For long-term storage, it is advisable to add a carrier protein such as HSA or BSA (0.1%) and avoid repeated freeze-thaw cycles.
The purity of this product is determined to be greater than 95.0% based on SDS-PAGE analysis.
The biological activity of this product is assessed based on its capacity to induce osteoclast formation in murine RAW264.7 cells. Using a concentration of 50ng/ml, this activity is demonstrated in a study titled "Corning® Osteo Assay Surface 24 Well Plates with Transwell® Permeable Supports- A Useful Tool for Co-Culture Studies" by Rebecca M. Wood and Mark Rothenber. This corresponds to a specific activity of 20,000 Units/mg.
PAMMEGSWLD VAQRGKPEAQ PFAHLTINAA SIPSGSHKVT LSSWYHDRGW AKISNMTLSN GKLRVNQDGF YYLYANICFR HHETSGSVPT DYLQLMVYVV KTSIKIPSSH NLMKGGSTKN WSGNSEFHFY SINVGGFFKL RAGEEISIQV SNPSLLDPDQ DATYFGAFKV QDID.
Q&A
What is sRANKL and how does it differ from membrane-bound RANKL in mice?
sRANKL is the soluble form of receptor activator of nuclear factor-κB ligand, a TNF-family cytokine essential for osteoclast formation. In mice, RANKL is initially produced as a type II transmembrane protein that can be cleaved by proteases to yield the soluble form (sRANKL) . The membrane-bound and soluble forms have distinct biological functions. Research has demonstrated that membrane-bound RANKL is sufficient for most physiological functions, including lymphocyte production, lymph node development, and mammary gland development, while the soluble form contributes specifically to physiological bone remodeling in adult mice .
What are the physiological roles of sRANKL in mouse bone metabolism?
In normal mouse physiology, sRANKL contributes to osteoclast formation and bone remodeling, particularly in adult mice. Studies have shown that:
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sRANKL binds to RANK on the surface of myeloid cells, stimulating their differentiation into osteoclasts
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The absence of soluble RANKL reduces osteoclast number and increases cancellous bone mass in adult mice, but not in growing mice
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Physiological bone remodeling in adult mice partially depends on soluble RANKL, whereas developmental processes do not require the soluble form
What mouse models are available for studying sRANKL function?
Several mouse models have been developed to study sRANKL function:
How can researchers establish a rapid bone loss model using recombinant sRANKL in mice?
Researchers can establish a rapid bone loss model by administering glutathione-S transferase-RANKL fusion protein (GST-RANKL) to mice . This approach offers several advantages:
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Rapid model development (within 50 hours) compared to traditional models that may take months
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Allows for quick evaluation of potential therapeutic compounds
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Produces significant and reproducible bone loss
The protocol involves:
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Preparation of purified GST-RANKL fusion protein (>95% purity, low endotoxin level of 0.01 ng/mg)
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Administration at appropriate dosage (determined by titration)
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Analysis of bone parameters after 2-7 days
This model enables researchers to rapidly evaluate anti-resorptive compounds, making it valuable for screening potential osteoporosis treatments .
What are the optimal methods for measuring sRANKL levels in mouse samples?
Measuring sRANKL levels in mouse samples requires sensitive and specific methodologies:
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ELISA-based detection: Commercially available ELISAs specifically designed for free mouse sRANKL provide the most reliable quantification in serum samples . These assays can distinguish between free sRANKL and sRANKL bound to osteoprotegerin (OPG).
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Plasma collection considerations: When collecting blood for sRANKL analysis, EDTA-treated plasma is preferred to prevent ex vivo degradation of the protein. Studies show that sRANKL levels in mouse plasma show low diurnal variability and are not significantly influenced by dietary factors .
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Tissue expression analysis: For tissue samples, immunohistochemistry using anti-RANKL antibodies can localize RANKL expression, while RT-PCR can quantify RANKL mRNA expression levels in different tissues.
How can researchers induce osteoclast formation using sRANKL in mouse cell cultures?
To induce osteoclast formation using sRANKL in vitro, researchers should follow these methodological approaches:
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Mouse bone marrow-derived macrophage (BMM) culture:
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Isolate bone marrow cells from mouse femurs and tibias
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Culture cells with macrophage colony-stimulating factor (M-CSF, 10,000 U/ml) for 2-3 days
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Add purified sRANKL at a final concentration of 5nM
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Culture for an additional 3 days
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Fix cells and perform TRAP (tartrate-resistant acid phosphatase) staining to identify multinucleated osteoclasts
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RAW264.7 cell culture:
The quality of recombinant sRANKL is critical; high purity (>95%) and low endotoxin levels (<0.01 ng/mg) are essential for reliable results .
How does sRANKL contribute to pathological conditions in mouse models of osteoarthritis?
In collagenase-induced osteoarthritis (CIOA) mouse models, sRANKL plays a significant role in disease progression:
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Early phase of CIOA:
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Late stage of CIOA:
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Systemic effects:
These findings suggest that increased local and systemic levels of sRANKL serve as biomarkers for osteoarthritis in mice, with potential translational implications for human OA .
What is the role of sRANKL in estrogen deficiency-induced bone loss in mice?
Despite the contribution of sRANKL to normal bone remodeling, research has revealed that:
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The bone loss caused by estrogen deficiency is unaffected by the lack of soluble RANKL in mouse models with sheddase-resistant RANKL
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This finding suggests that membrane-bound RANKL is sufficient to mediate estrogen deficiency-induced bone loss, whereas soluble RANKL contributes primarily to physiological bone remodeling in adult mice
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The distinction between these pathways has important implications for therapeutic targeting of RANKL in postmenopausal osteoporosis, suggesting that inhibition of both forms may be necessary for complete prevention of bone loss
How do the effects of sRANKL differ between growing and adult mice?
Research has uncovered significant age-dependent variations in sRANKL effects:
These findings suggest that sRANKL plays a more significant role in adult bone homeostasis than in developmental bone formation, with implications for age-specific therapeutic approaches .
How can researchers differentiate between the effects of sRANKL and membrane-bound RANKL in mouse studies?
To differentiate between the effects of soluble and membrane-bound RANKL, researchers can employ several methodological approaches:
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Use of sheddase-resistant RANKL mouse models: These mice express normal levels of membrane-bound RANKL but have undetectable levels of circulating soluble RANKL, allowing for the isolation of membrane-bound RANKL functions
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Administration of recombinant sRANKL: Direct administration of purified sRANKL to mice allows researchers to study effects specifically attributable to the soluble form
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Form-specific neutralizing antibodies: When available, antibodies that specifically neutralize either the soluble or membrane-bound form can help distinguish their relative contributions
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Tissue-specific gene deletion: Conditional knockout of RANKL in specific cell types can help identify the cellular sources of physiologically relevant sRANKL
This differential analysis is critical for understanding which pathological processes require intervention targeting specific RANKL forms .
What are the critical storage and handling requirements for recombinant sRANKL in mouse experiments?
Proper storage and handling of recombinant sRANKL is essential for maintaining its biological activity:
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Storage temperature: sRANKL must be stored at ultra-low temperatures (-70°C or below) to maintain activity
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Freeze-thaw cycles: Avoid unnecessary freeze-thaw cycles as they can significantly reduce biological activity
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Buffer considerations: sRANKL is typically stored in PBS with 1 mM EDTA to maintain stability
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Concentration: Working concentrations should be determined empirically for each application, but 5nM is commonly used for osteoclast formation assays
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Quality control: Prior to use, verify protein purity (>95% by SDS-PAGE) and ensure low endotoxin levels (<0.01 ng/mg) to prevent confounding inflammatory responses
How can researchers resolve common issues with sRANKL-induced osteoclastogenesis in mouse cell cultures?
When troubleshooting sRANKL-induced osteoclastogenesis experiments, researchers should consider:
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Recombinant protein quality: Ensure the sRANKL protein is of high purity and properly folded. Compare results with validated commercial sources if inconsistencies are observed
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Cell source variation: Bone marrow-derived macrophages from different mouse strains may respond differently to sRANKL stimulation. Standardize the strain, age, and sex of mice used for cell isolation
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M-CSF priming: Proper priming of myeloid precursors with M-CSF (10,000 U/ml) is essential before sRANKL addition
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Culture duration optimization: The optimal duration for osteoclast formation is typically 3-4 days after sRANKL addition, but this may vary based on experimental conditions
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TRAP staining technique: For reliable identification of mature osteoclasts, ensure proper fixation and staining procedures, focusing on multinucleated (≥3 nuclei) TRAP-positive cells
What emerging approaches are being developed for studying sRANKL function in mouse models?
Several innovative approaches are emerging for studying sRANKL function:
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CRISPR-Cas9 gene editing: Creation of more precise mouse models with specific modifications to RANKL cleavage sites or signaling domains
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Tissue-specific and inducible RANKL modification: Development of conditional mouse models that allow temporal control of RANKL shedding in specific tissues
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Bispecific antibodies: Development of antibodies that can distinguish between different forms of RANKL for more targeted research
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Advanced imaging techniques: Application of intravital imaging to visualize RANKL-RANK interactions in bone microenvironments in real-time
These approaches promise to provide more nuanced understanding of sRANKL biology and may reveal new therapeutic targets for bone disorders.
How might findings from mouse sRANKL research translate to human clinical applications?
Translational aspects of mouse sRANKL research include:
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Biomarker development: Elevated sRANKL levels observed in mouse models of osteoarthritis correlate with similar findings in human patients, suggesting potential diagnostic applications
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Therapeutic targeting: The development of denosumab, a fully human anti-RANKL monoclonal antibody, was informed by mouse model research and is now clinically used for treating osteoporosis and cancer-related bone disorders
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Differential targeting: Understanding the distinct roles of soluble versus membrane-bound RANKL in mice may inform more selective therapeutic approaches in humans
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Rapid screening platforms: Mouse models of rapid bone loss via sRANKL administration provide efficient platforms for evaluating potential therapeutic compounds before human trials
The continued refinement of mouse models will likely lead to more precise interventions for human bone disorders in the future.
Product Science Overview
RANKL is a type II transmembrane protein with an extracellular domain at the carboxy-terminus . This ectodomain can be cleaved by enzymes such as matrix metalloproteinases, releasing a soluble form of RANKL into the extracellular environment . The soluble form of RANKL is often used in research and therapeutic applications due to its ability to interact with its receptor, RANK, on the surface of target cells.
RANKL is expressed in several tissues and cell types, including fibroblasts, T cells, and osteoblasts . It plays a significant role in the differentiation and activation of osteoclasts, which are cells responsible for bone resorption . Additionally, RANKL is involved in the maturation of dendritic cells and the regulation of T cell-dependent immune responses .
The primary function of RANKL is to bind to its receptor, RANK, which is expressed on the surface of osteoclast precursors and mature osteoclasts . This binding triggers a signaling cascade that leads to the differentiation, activation, and survival of osteoclasts . Osteoclasts are essential for bone remodeling, a process that maintains bone health by balancing bone formation and resorption.
RANKL also interacts with osteoprotegerin (OPG), a decoy receptor that inhibits the binding of RANKL to RANK . This interaction prevents osteoclastogenesis and leads to the accumulation of bone and cartilage . High expression of RANKL is observed during degenerative bone diseases and in tumors, highlighting its role in pathological conditions .
Recombinant mouse RANKL, particularly its soluble form, is widely used in research to study bone metabolism, immune cell activation, and tumor growth . It is also utilized in differentiation studies and functional assays involving osteoclasts and other cell types . The recombinant protein is typically produced in bacterial expression systems, such as Escherichia coli, and is purified to high standards for use in various experimental applications .
