Recombinant Mouse Kit ligand protein (Kitlg), partial (Active)

What is the basic function of Recombinant Mouse Kit Ligand Protein in mammals?

Recombinant Mouse Kit Ligand Protein (Kitlg), also known as Stem Cell Factor (SCF), is a cytokine that binds to the receptor tyrosine kinase Kit (c-kit). This interaction plays crucial roles in multiple biological processes including hematopoiesis, melanogenesis, gametogenesis, and neuronal development. At the molecular level, noncovalent dimers of transmembrane or soluble SCF interact with the receptor tyrosine kinase SCF R/c-kit to trigger receptor dimerization and signaling . In the nervous system, Kit is enriched in molecular layer interneurons (MLIs) of the cerebellar cortex, while Kit Ligand is selectively expressed by Purkinje cells (PCs) . This reciprocal expression pattern is functionally significant as genetic manipulations have demonstrated that PC Kit Ligand and MLI Kit are required for proper inhibition of PCs . Beyond these regulatory functions, Kit Ligand also plays a role in energy metabolism and contributes to temperature adaptation in mammals .

What are the species-specific characteristics of Kit Ligand in mice compared to other mammals?

Mouse Kit Ligand shows specific sequence identity patterns with other species that affect its cross-species reactivity. Within the extracellular domain (ECD) of the short isoform, mouse SCF shares 93% amino acid sequence identity with rat SCF and 72-75% with canine, feline, and human SCF . These sequence differences translate to important functional variations in cross-species activity.

In evolutionary studies, KITLG has shown evidence of Darwinian positive selection in both European and East Asian human populations, with different variants being selected for different adaptive traits . While this selection mainly occurred in human populations, it demonstrates the evolutionary importance of this gene across mammals.

How does Recombinant Mouse Kit Ligand Protein influence neuronal function?

Recombinant Mouse Kit Ligand Protein plays a significant role in neuronal function, particularly in the cerebellum. Research has demonstrated that the Kit Ligand/Kit receptor dyad sustains mammalian central synapse function. The expression pattern of these proteins is mutually exclusive and cell-type specific: Kit is enriched in molecular layer interneurons (MLIs) of the cerebellar cortex (stellate and basket cells), while Kit Ligand is selectively expressed by Purkinje cells (PCs) .

Functional studies using in vivo genetic manipulation spanning from embryonic development through adulthood have shown that PC Kit Ligand and MLI Kit are required for proper inhibition of PCs. These proteins are not only necessary for maintaining this inhibition but are also capable of driving changes in it .

This neuron-specific Kit Ligand/Kit receptor interaction contributes to proper synaptic function in the mammalian brain. Disruptions in this system have been linked to neurodevelopmental disorders, suggesting its importance in proper brain development and function .

What are the optimal conditions for evaluating bioactivity of Recombinant Mouse Kit Ligand Protein in vitro?

To evaluate the bioactivity of Recombinant Mouse Kit Ligand Protein, the TF-1 human erythroleukemic cell line proliferation assay has been established as a reliable method. In standardized protocols, the effective dose (ED50) for stimulating cell proliferation ranges from 2.5-10 ng/mL . When conducting this assay, researchers should consider the following methodological aspects:

Cell preparation is crucial - TF-1 cells should be maintained in optimal growth conditions prior to the assay and should be in the exponential growth phase. A proper dose-response testing protocol should include a range of concentrations (typically 0.1-100 ng/mL) to establish the full dose-response curve for each batch of recombinant protein. Standard incubation conditions include 37°C with 5% CO2 in a humidified incubator, with an incubation period of 48-72 hours.

Cell proliferation can be quantified using colorimetric assays such as MTT or MTS, or by direct cell counting. Each experiment should include positive controls (known active Kit Ligand) and negative controls (buffer without Kit Ligand) to validate assay performance.

When evaluating temperature-dependent effects, special consideration should be given to experimental conditions. Research has shown that KITLG has pleiotropic effects related to both pigmentation and thermogenesis. For in vitro functional tests at low temperature, specific protocols have been developed to assess the adaptive response related to winter temperature .

How can researchers effectively design experiments to study the dual roles of Kit Ligand in pigmentation and thermal adaptation?

Designing experiments to study the dual roles of Kit Ligand in pigmentation and thermal adaptation requires a multifaceted approach that addresses both functions simultaneously while controlling for confounding variables. Based on current research findings , the following experimental design framework is recommended:

For genotype-phenotype correlation studies, researchers should select populations with known KITLG variants, particularly those showing evidence of selection in the upstream and downstream regions. Measurements should include both pigmentation phenotypes (skin reflectance, melanin index) and thermogenesis markers (basal metabolic rate, cold-induced thermogenesis). Statistical methods can then be applied to detect associations between specific KITLG variants and each phenotype.

In vitro functional validation should involve enhancer reporter assays to test the activity of selected KITLG variants under different temperature conditions. Cell lines relevant to both pigmentation (melanocytes) and thermogenesis (brown adipocytes) should be used, and cells should be subjected to temperature gradients (37°C, 32°C, and 27°C) to assess temperature-dependent responses.

Transgenic mouse models carrying specific human KITLG variants of interest can be used to evaluate both pigmentation changes and thermal adaptation metrics. Cold challenge tests (4-8°C) can assess thermogenic capacity, while molecular analyses can examine changes in both melanocytes and thermogenic tissues.

Geographic correlation analyses can compare allele frequencies of KITLG variants with both UV radiation index and winter temperature data across populations. Partial correlation and multiple regression analyses can separate the effects of UV and temperature.

Research has demonstrated that different KITLG sequence variants are targeted by natural selection for different adaptive purposes, with some variants showing stronger correlation with winter temperature than with UV radiation in Asian populations .

What methods are most effective for studying Kit Ligand/Kit receptor interactions in neural tissues?

Studying Kit Ligand/Kit receptor interactions in neural tissues requires specialized techniques that preserve the integrity of neural circuits while allowing for precise manipulation and measurement. Based on recent research , the following methodological approaches are recommended:

Genetic manipulation strategies should include conditional knockout models that allow temporal control (from embryonic development through adulthood) and cell-type specific manipulations targeting Kit in molecular layer interneurons (MLIs) and Kit Ligand in Purkinje cells (PCs). The use of Cre-loxP systems with neural-specific promoters enables precise genetic targeting.

Electrophysiological recordings, such as whole-cell patch-clamp recordings of Purkinje cells, can measure inhibitory postsynaptic currents (IPSCs). Paired-pulse stimulation protocols with stimulating electrodes placed in the outer or inner molecular layer allow analysis of parameters such as IPSC amplitude and paired pulse ratio to assess synaptic function.

Cellular characterization should assess neuronal capacitance, input resistance, and spontaneous or evoked action potential frequency. Quantification of cell numbers and distribution using immunohistochemistry and analysis of evoked neurotransmitter release through direct electrode stimulation provide additional insights.

Molecular signaling analysis can investigate downstream signaling cascades activated by Kit receptor tyrosine kinase, assess protein phosphorylation states using phospho-specific antibodies, and evaluate gene expression changes in response to Kit Ligand/Kit receptor signaling.

Recent research has shown that despite normal MLI numbers, distribution, and firing patterns in Kit knockout models, there are defects in PC inhibition, suggesting specific effects on synaptic function rather than neuronal development or excitability .

How do genetic variants in the KITLG gene influence Kit Ligand function and adaptation?

Genetic variants in the KITLG gene have been extensively studied for their influence on Kit Ligand function and their role in human adaptation. Multiple selection tests have revealed strong signals of positive selection in both European and East Asian populations, particularly in the regulatory regions surrounding the gene .

The selection signals are detected in different regions of the KITLG locus in different populations. In the upstream region (126-375 kb upstream of KITLG), strong selection signals are detected in Europeans by the EHH test, with relatively weaker signals in East Asians . This region contains enhancer elements that regulate KITLG expression, and variants in this region (e.g., rs642742 and rs12821256) significantly affect skin color in African-Americans and hair color in Europeans .

In the downstream region (57.5 kb downstream of KITLG), a selection signal is observed specifically in East Asians, confirmed by the XP-CLR test using Africans as an outgroup . The variant rs6538148 in this region has been associated with mean corpuscular hemoglobin concentration and red blood cell distribution in Europeans .

Functional analyses have demonstrated that the selected adaptive alleles in East Asians cause reduced enhancer activity, which presumably leads to reduced expression of KITLG. This reduction influences melanocyte development and results in skin lightening .

Interestingly, different KITLG variants show adaptation to different environmental factors. While some variants are associated with adaptation to weak UV radiation at high latitudes, others show stronger correlation with winter temperature, especially in Asian populations .

What methodologies are most effective for detecting selective sweeps in the KITLG gene across different populations?

Detecting selective sweeps in the KITLG gene across different populations requires a comprehensive approach using multiple complementary methods. Based on the research methodologies described in the literature , the following approaches are most effective:

Haplotype-based methods include the Extended Haplotype Homozygosity (EHH) test, which detects extended homozygosity surrounding variants under positive selection. This test is sensitive to relatively recent selection (<100,000 years) . Cross-population Extended Haplotype Homozygosity (XP-EHH) compares haplotype lengths between populations to identify population-specific selective sweeps. The Integrated Haplotype Score (iHS) identifies selected alleles that have increased in frequency but haven't yet reached fixation.

Allele frequency spectrum-based methods such as Tajima's D test, Fu and Li's test, and Fay and Wu's H test examine different aspects of the allele frequency distribution to detect signatures of selection. Population differentiation methods like XP-CLR (Cross-population Composite Likelihood Ratio) identify highly differentiated genomic regions as targets of selective sweeps, with each spot in the analysis representing the average value of a 2-kb window .

For KITLG analysis, examining a large genomic region (e.g., 549 kb, covering both upstream and downstream noncoding regions) is recommended . Each test should use appropriate window sizes (e.g., 3-kb windows for EHH test, 2-kb windows for XP-CLR test).

Evolutionary reconstruction methods can include maximum-likelihood tree construction using RAxML with sequence variants with allele frequency (MAF) ≥0.05 . Comparison with homologous sequences from chimpanzee, Neanderthal, and Denisovan provides evolutionary context.

Selection onset time estimation using Hidden Markov Model (HMM) approaches can infer selection onset time by analyzing haplotype structure around putatively selected SNP loci . Bootstrapping over haplotypes helps achieve confidence intervals for these estimates.

These methods have successfully identified recurrent selective events in both the upstream and downstream regions of KITLG in Eurasian populations, demonstrating adaptation to both UV radiation and winter temperature at high latitude areas .

How can researchers effectively analyze the pleiotropic effects of Kit Ligand in different physiological systems?

Analyzing the pleiotropic effects of Kit Ligand across different physiological systems requires an integrated approach that combines multiple research methodologies and considers the diverse functions of this protein. Based on the current understanding of Kit Ligand's roles in pigmentation, thermogenesis, and neural function , the following research framework is recommended:

Comparative genomic and functional analysis should identify conserved and divergent regulatory elements across species, compare expression patterns of KITLG in different tissues (skin, neural, hematopoietic, reproductive), and analyze tissue-specific enhancers and their responsiveness to different environmental stimuli.

A systems biology approach can construct gene regulatory networks centered around KITLG in different cell types, identify shared and distinct signaling pathways activated by Kit Ligand/Kit receptor interaction, and use transcriptomic and proteomic analyses to map downstream effects in different tissues.

Conditional manipulation experiments should develop tissue-specific and temporally controlled knockout or overexpression models, assess phenotypic changes across multiple physiological systems simultaneously, and use CRISPR-Cas9 to introduce specific variants associated with different adaptive traits.

Environmental response studies can subject model organisms with KITLG variants to different environmental conditions (UV exposure, temperature variation), measure physiological responses across multiple systems (skin pigmentation, energy expenditure, neural function), and analyze gene-environment interactions using statistical methods that account for pleiotropic effects.

Clinical correlation studies can investigate associations between KITLG variants and multiple phenotypes in human populations, analyze comorbidities of conditions linked to KITLG dysfunction, and develop multivariate models to assess the contribution of KITLG to different phenotypes.

Research has demonstrated that KITLG exhibits pleiotropic effects in pigmentation and thermogenesis, with different variants being targeted by natural selection for adaptation to both UV radiation and winter temperature in Eurasian populations . Similarly, in the nervous system, Kit Ligand/Kit receptor interactions play specific roles in maintaining synaptic function between cerebellar neurons .

What are the critical quality control parameters for Recombinant Mouse Kit Ligand Protein?

Ensuring the quality and consistency of Recombinant Mouse Kit Ligand Protein is crucial for reliable experimental results. Based on industry standards and the literature , the following quality control parameters are critical:

Purity assessment using SDS-PAGE analysis under reducing conditions should show a single band at approximately 19 kDa . Silver staining techniques provide high sensitivity for detecting protein contaminants, and purity should be >95% as determined by densitometric analysis.

Bioactivity testing using cell proliferation assay with TF-1 human erythroleukemic cell line should be performed to determine ED50 (effective dose at 50% maximum response), which should fall within the range of 2.5-10 ng/mL . Dose-response curves should demonstrate appropriate sigmoidal shape.

Protein structure verification should confirm proper folding using circular dichroism or other spectroscopic methods, assess noncovalent dimer formation (which is essential for receptor activation), and verify critical post-translational modifications.

Endotoxin testing using Limulus Amebocyte Lysate (LAL) assay ensures endotoxin levels are below acceptable limits, typically <1.0 EU/μg of protein. Stability assessment should include accelerated stability studies at different temperatures, freeze-thaw stability to ensure protein maintains activity after typical laboratory handling, and long-term storage stability at recommended conditions (-20°C to -80°C).

Batch consistency comparison is essential, with a Certificate of Analysis (CoA) for each production batch documenting all quality control results. Any significant deviation from the expected molecular weight (19 kDa) or the presence of additional bands may indicate degradation or contamination .

How can researchers optimize the expression and purification of functional Recombinant Mouse Kit Ligand?

Optimizing the expression and purification of functional Recombinant Mouse Kit Ligand requires careful consideration of multiple factors throughout the production process. Based on standard recombinant protein production protocols and specific considerations for Kit Ligand , the following optimization strategies are recommended:

Expression system selection is critical - mammalian expression systems (CHO, HEK293) are preferred for proper folding and post-translational modifications. Insect cell systems (Sf9, High Five) can provide higher yields with mammalian-like glycosylation, while E. coli systems may be used for non-glycosylated variants but require refolding protocols.

Vector design considerations should include optimized codon usage for the expression host, appropriate secretion signal sequences for extracellular production, and fusion tags that can be removed without affecting protein function.

Expression optimization should involve small-scale expression trials to determine optimal induction timing and duration, cell density at induction, media composition and supplements, and temperature during expression phase (often reduced to 30-32°C).

A multi-step purification strategy typically includes affinity chromatography (His-tag, GST-tag, or immunoaffinity), ion exchange chromatography, and size exclusion chromatography for final polishing and buffer exchange.

Protein stability considerations require identifying and optimizing buffer conditions (pH, ionic strength, additives), considering the inclusion of stabilizing agents (glycerol, specific sugars), and determining optimal concentration for storage to prevent aggregation.

Specific considerations for Kit Ligand include expression of the proper isoform (mouse SCF has an alternatively spliced isoform lacking 28 amino acids that encompasses the primary proteolytic recognition site) , attention to the extracellular domain (ECD) structure (which is critical for bioactivity), and monitoring of proteolytic processing.

The recombinant mouse Kit Ligand should be evaluated not only for purity but also for functional activity, particularly its ability to stimulate cell proliferation in appropriate bioassays with an ED50 in the range of 2.5-10 ng/mL .

What analytical methods best characterize the structural and functional properties of Recombinant Mouse Kit Ligand Protein?

Comprehensive characterization of Recombinant Mouse Kit Ligand Protein requires a combination of analytical methods that assess both structural integrity and functional activity. Based on standard protein characterization approaches and specific considerations for Kit Ligand , the following analytical methods are recommended:

For primary structure analysis, mass spectrometry (LC-MS/MS) confirms sequence and maps post-translational modifications, N-terminal sequencing verifies correct processing of the signal peptide, and peptide mapping ensures complete sequence coverage. Secondary and tertiary structure analysis can employ circular dichroism (CD) spectroscopy to assess secondary structure elements, Fourier-transform infrared spectroscopy (FTIR) for complementary secondary structure information, and intrinsic fluorescence spectroscopy to monitor tertiary structure.

Quaternary structure analysis methods include size-exclusion chromatography with multi-angle light scattering (SEC-MALS) to determine molecular weight and oligomeric state, analytical ultracentrifugation to assess heterogeneity and confirm noncovalent dimer formation, and native mass spectrometry for intact complex analysis.

Functional characterization should include receptor binding assays such as surface plasmon resonance (SPR) to determine binding kinetics and affinity to Kit receptor, bio-layer interferometry (BLI) as an alternative label-free binding analysis, and cell-based receptor binding assays using Kit-expressing cells. Bioactivity assays should include TF-1 cell proliferation assay with dose-response analysis (ED50 determination) , phosphorylation assays to monitor Kit receptor activation and downstream signaling, and cell migration assays to assess chemotactic activity.

Specialized functional tests can include temperature-dependent activity assays to assess thermogenic functions , melanocyte development assays to evaluate effects on pigmentation, and electrophysiological measurements for neural function (particularly for cerebellar studies) .

The combination of SDS-PAGE analysis showing a single band at 19 kDa under reducing conditions and bioactivity testing in TF-1 cells with an ED50 of 2.5-10 ng/mL provides the foundation for basic characterization. More sophisticated analyses provide deeper insights into structural and functional properties.

How can Recombinant Mouse Kit Ligand be utilized in neural developmental studies?

Recombinant Mouse Kit Ligand offers significant potential for neural developmental studies, particularly in understanding synaptic formation and function. Based on recent research demonstrating the role of Kit Ligand/Kit receptor signaling in cerebellar circuits , the following applications are recommended:

For investigating synapse development and maintenance, researchers can use recombinant Kit Ligand in primary neuronal cultures to assess effects on synapse formation, apply the protein at different developmental timepoints to determine critical periods for Kit signaling, and combine with electrophysiological recordings to measure functional changes in synaptic strength.

Cerebellar circuit developmental studies can utilize organotypic slice cultures of developing cerebellum treated with recombinant Kit Ligand, assess effects on molecular layer interneuron (MLI) migration, differentiation, and connectivity, and investigate the reciprocal expression pattern between Kit Ligand in Purkinje cells and Kit in MLIs .

For neurodevelopmental disorder modeling, researchers can apply recombinant Kit Ligand to neural stem cell cultures from models of neurodevelopmental disorders, assess rescue effects on cellular phenotypes associated with these disorders, and investigate how Kit Ligand signaling interacts with other pathways implicated in neurodevelopmental conditions.

Methodological approaches should include time-lapse imaging of neurons in the presence of recombinant Kit Ligand to track morphological changes, whole-cell patch-clamp recordings to measure electrophysiological responses , calcium imaging to assess changes in neuronal activity patterns, and molecular profiling of Kit Ligand-responsive neurons to identify downstream effectors.

Research has demonstrated that the Kit Ligand/Kit receptor dyad sustains mammalian central synapse function, particularly in the cerebellum where Kit Ligand expressed by Purkinje cells signals to Kit receptors on molecular layer interneurons . This system provides an excellent model for studying cell-type specific ligand-receptor interactions in neural development and function.

What are the emerging research areas linking Kit Ligand function to adaptive physiological responses?

Emerging research has uncovered novel roles for Kit Ligand in adaptive physiological responses beyond its well-established functions in development and stem cell maintenance. Based on recent findings , several cutting-edge research areas are developing:

Thermal adaptation mechanisms research is investigating Kit Ligand's role in thermogenesis and energy expenditure, analyzing how KITLG variants affect cold adaptation through mitochondrial function, and studying brown adipose tissue activation in response to Kit Ligand signaling.

Integrated environmental adaptation studies focus on how KITLG variants simultaneously affect multiple adaptive traits, investigating pleiotropic effects across different physiological systems, and analyzing the molecular mechanisms underlying adaptation to both UV radiation and temperature .

Temporal dynamics of adaptation research studies the evolutionary timing of selection on different KITLG variants, analyzes how recent climate changes might affect selection pressure on KITLG, and investigates ongoing selection in contemporary human populations.

Mechanistic studies of pleiotropy research examines how the same gene can affect different physiological systems, analyzes tissue-specific enhancers and their responsiveness to different environmental stimuli, and investigates downstream signaling pathways that differ between tissues.

Research has demonstrated that the pleiotropic effects of KITLG, specifically pigmentation and thermogenesis, were both targeted by natural selection that acted on different KITLG sequence variants, contributing to the adaptation of Eurasians to both UV radiation and winter temperature at high latitude areas . This finding opens new avenues for research on the molecular mechanisms of adaptation and the role of pleiotropy in evolution.

What methodological approaches can integrate Kit Ligand research across different physiological systems?

Integrating Kit Ligand research across different physiological systems requires interdisciplinary approaches that can capture the protein's diverse functions while identifying common mechanisms. Based on current research spanning neural , pigmentation, and thermal adaptation systems , the following methodological approaches are recommended:

Multi-omics integration strategies include parallel transcriptomic, proteomic, and metabolomic analyses across tissues affected by Kit Ligand, network biology approaches to identify shared and distinct signaling pathways, and machine learning methods to predict functional consequences of KITLG variants across systems.

Comparative physiological approaches involve cross-species analysis of Kit Ligand function in model organisms adapted to different environments, measurement of multiple physiological parameters (pigmentation, thermogenesis, neural function) in the same experimental subjects, and development of integrated physiological assessment protocols for Kit Ligand function.

Advanced genetic approaches can include CRISPR-based enhancer scanning to identify regulatory elements controlling tissue-specific expression, parallel reporter assays to simultaneously test multiple variants in different cell types, and tissue-specific conditional knockouts with temporal control to isolate system-specific functions.

Innovative cellular models can involve co-culture systems combining different cell types that express Kit or Kit Ligand, organoid models representing different physiological systems affected by Kit Ligand, and microfluidic platforms to study cell-cell interactions mediated by Kit Ligand signaling.

Research has demonstrated that KITLG exhibits pleiotropic effects, influencing both pigmentation in response to UV radiation and thermogenesis in adaptation to winter temperature , while also playing critical roles in neural function, particularly in cerebellar circuits . An integrated research approach can elucidate how these diverse functions are coordinated at the molecular level and how they have been shaped by evolution.