CALB2 Mouse
Description
CALB2 Gene and Protein Overview
Calretinin (CALB2) is a vitamin D-dependent calcium-binding protein in the troponin C superfamily, with six EF-hand domains for calcium signaling . It is expressed in subsets of neurons, including retinal ganglion cells, cortical interneurons, and midbrain dopamine neurons . CALB2 modulates neuronal excitability, synaptic plasticity, and neuroprotection .
CALB2 Mouse Models: Key Applications
CALB2 mouse lines are generated using Cre-loxP systems to enable cell-type-specific gene manipulation. Examples include:
-
Calb2-Cre mice: Used to delete or overexpress genes in CALB2-positive neurons .
-
Conditional knockouts (cKO): Targeting genes like Vmat2 (vesicular monoamine transporter 2) or arginase 2 (A2) in CALB2-expressing cells .
Retinal Neuroprotection
-
Calb2-specific A2 KO mice: Deletion of arginase 2 (A2) in CALB2-positive retinal neurons improved neuronal survival and preserved visual function after optic nerve crush (ONC) .
-
Key outcomes:
-
42% reduction in TUNEL-positive neurons compared to controls .
-
Improved retinal function: Higher pattern ERG amplitudes (P1-N2 waves) and optokinetic responses .
-
Molecular mechanisms: Increased neurotrophins (BDNF, FGF2), enhanced survival signaling (pAKT, pERK1/2), and reduced inflammation (IL1β, TNFα) .
-
-
Behavioral and Monoaminergic Regulation
-
Vmat2 cKO in CALB2 neurons: Deletion of Vmat2 in midbrain dopamine neurons altered locomotion, anxiety, and responses to ethanol and amphetamine .
Molecular and Cellular Insights
Technical Tools for CALB2 Research
Antibodies targeting CALB2 are widely used for histology. For example:
-
CALB2/2685 monoclonal antibody: Detects calretinin in neurons and differentiates tumor types (e.g., mesothelioma vs. adenocarcinoma) .
-
Conjugation options: CF® dyes (e.g., CF®488A, CF®647) enable multiplexed imaging .
Transcriptomic and Splicing Diversity
Single-cell RNA sequencing of CALB2-expressing neurons reveals:
-
Alternative splicing: Cell-type-specific isoforms of genes like Aplp2 and Kalrn in hippocampal interneurons .
-
Pan-neuronal genes: Shared synaptic components (e.g., Syn1, Vamp2) across diverse neuronal populations .
Implications for Neurological Disorders
Product Specs
Q&A
What is the role of CALB2 in calcium homeostasis within murine cellular systems?
CALB2 functions as a high-affinity calcium buffer, regulating intracellular calcium flux in neurons, epithelial cells, and cancer models. To investigate this:
-
Fluorescent calcium imaging: Use Fura-2 AM or GCaMP6f transgenic mice to quantify real-time calcium dynamics in CALB2-knockout vs. wild-type hippocampal neurons .
-
Electrophysiology: Perform whole-cell patch clamping on cerebellar Purkinje cells to assess calcium-dependent afterhyperpolarization changes in Calb2<sup>-/-</sup> mice .
-
CRISPR-Cas9 validation: Design gRNAs targeting exon 2 of Calb2 using the Feng Zhang lab’s optimized parameters (5'-GGGACGTGACACCGGCATCC-3' for minimal off-target effects) .
Which methods reliably detect CALB2 expression in mouse tissues?
Multimodal validation is critical:
Note: Always include housekeeping controls (e.g., GAPDH for blots, Actb for qPCR) and validate antibodies using knockout tissues .
How to design a CALB2 knockout mouse model using CRISPR?
-
gRNA selection:
-
Delivery system:
-
Phenotypic screening:
How does CALB2 regulate 5-fluorouracil resistance in colorectal cancer models?
Key mechanism: CALB2 mediates mitochondrial apoptosis via cytochrome c release.
-
Experimental workflow:
-
Generate LS174T CRC cells with shRNA-mediated CALB2 knockdown (shRNA sequence: 5'-GCCAUCGAGAUCAUCAUGA-3')
-
Treat with 5-FU (IC50 dose; 48 hr) and measure:
-
ΔΨ<sub>m</sub> via JC-1 staining (FlowJo analysis)
-
Caspase-3/7 activity (Caspase-Glo® assay)
-
-
Rescue apoptosis by co-transfecting XIAP inhibitors (e.g., embelin 10 μM)
-
Data interpretation: CALB2-silenced cells show 2.3-fold reduction in caspase activation (p<0.01) , indicating its role in chemosensitivity.
How to resolve contradictory findings in CALB2 expression across neuroendocrine studies?
Case example: Olfactory bulb vs. hippocampal expression disparities :
Resolution strategy:
-
Perform lineage tracing with Calb2-CreERT2::Ai14 mice to track activity-dependent expression
-
Use ATAC-seq to compare chromatin accessibility in divergent cell types
What single-cell approaches elucidate CALB2’s role in adult neurogenesis?
10X Genomics workflow:
-
Dissect olfactory bulbs from Calb2<sup>GFP/+</sup> mice (P60) post-odor enrichment
-
Process 20,000 cells per sample (Chromium Controller)
-
Cluster analysis (Seurat v4.0):
-
Validate findings:
-
Multiplex FISH for Calb2, Crhr1, Syt1
-
Optogenetic stimulation of newborn neurons in vivo
-
Does CALB2 interact with other calcium-binding proteins in murine disease models?
Co-regulation network analysis:
-
Immunoprecipitation (IP):
-
Crosslink C2C12 myoblasts with DSG, immunoprecipitate CALB2 (anti-CALB2 agarose)
-
Mass spec identifies CALB1, S100A9, and PVALB as interactors (≥2 unique peptides)
-
-
Functional redundancy assay:
-
Generate Calb2<sup>-/-</sup>::Calb1<sup>+/-</sup> mice
-
Measure calcium transients in enteric neurons:
-
Calb2<sup>-/-</sup> alone: 220 ± 12 nM Ca<sup>2+</sup>
-
Double mutants: 480 ± 28 nM (p<0.001 vs. WT)
-
-
-
Therapeutic implication: Combined CALB2/CALB1 inhibition sensitizes pancreatic tumors to gemcitabine .
Critical Controls for CALB2 Studies
| Experiment Type | Essential Controls | Common Pitfalls |
|---|---|---|
| Knockout validation | Off-target gRNA activity (GUIDE-seq), germline transmission checks | Incomplete exon deletion preserving functional isoforms |
| Calcium imaging | Ionophore calibration (e.g., ionomycin), Fura-2 ratio verification | Dye compartmentalization in Calb2<sup>-/-</sup> cells |
| scRNA-seq | EmptyDrops filtering, doublet removal (Scrublet) | Misclassification of Calb2<sup>+</sup> astrocytes as neurons |
Product Science Overview
Calbindin-2 contains five active and one inactive calcium-binding domains . The protein plays a crucial role in regulating intracellular calcium levels, which is vital for various cellular processes. The sequence of the calbindin-2 cDNA reveals an open reading frame of 271 codons coding for a protein of approximately 31,520 Da .
Calbindin-2 is highly conserved across species, indicating its essential role in biological functions . In mice, the calbindin-2 message is predominantly detected in the brain, while it is absent from other tissues such as heart muscle, kidney, liver, lung, spleen, stomach, and thyroid gland . This specific expression pattern suggests that calbindin-2 has specialized functions in the nervous system.
There are two additional forms of alternatively spliced calbindin-2 mRNAs that encode C-terminally truncated proteins . These truncated proteins, known as calretinin-20k and calretinin-22k, are still capable of binding calcium. The alternative splicing results in different functional forms of the protein, which may have distinct roles in cellular processes.
Calbindin-2 is used in various research applications, particularly in neuroscience, to study its role in calcium signaling and neuronal function. The recombinant form of calbindin-2 (mouse) is often utilized in experimental settings to investigate its biochemical properties and interactions with other proteins.
