R26-CAG-lsl-CaMPARI2 mice express CaMPARI2 fluorescent calcium reporter from a CAG promoter introduced to the Gt(ROSA)26Sor gene when a loxP-flanked STOP cassette is excised with Cre recombinase. CaMPARI changes from green to red fluorescence with high intracellular calcium and photoconversion light, and is thus useful in monitoring neuronal activity. A poster describing the strain can be found here.
Dr. Mario R Capecchi, University of Utah
Dimitri Tränkner, University of Utah
CaMPARI (Calcium Modulated Photoactivatable Ratiometric Integrator) is a photoconvertible protein construct that enables imaging of the integrated calcium activity of large populations of cells over defined time windows. It is derived from the stony coral protein EOS, which irreversibly shifts its fluorescence. It photoconverts from green to red in the presence of UV light (~400 nm). By fusing calcium binding calmodulin and a calmodulin/Ca2+ binding peptide (M13) to either end, CaMPARI photoconversion becomes calcium dependent. This system enables the marking of active neuronal populations with single-cell and subsecond resolution. As such, neuronal ensembles can be functionally marked with high spatiotemporal resolution.
CaMPARI2, as compared to the original CaMPARI (CaMPARI1) construct, incorporates enhanced calcium binding affinity variants (CaMPARI1_W391F-V398L_Q142V-F198Y-C202T-L217I-N345S). These mutations were targeted around the fluorescent protein chromophore and at the protein interface between the fluorescent protein and the calcium-sensitive domains.
These R26-CAG-lsl-CaMPARI2 mice express CaMPARI2 from a CAG promoter introduced to the Gt(ROSA)26Sor gene when a loxP-flanked Stop cassette is excised with Cre recombinase. A poster describing the strain can be found here.
Though designed as an improved sensor with brighter fluorescence and faster calcium unbinding kinetics as compared to the original CaMPARI1 construct, fluorescence intensity in the R26-CAG-lsl-CaMPARI2 mouse does not quite reach the levels measured in vitro or in other model systems. Crosses with Emx1-cre produce heterozygous progeny with relatively weak green and red fluorescence in the cortex (2.5 fold background max). This may be higher in homozygous mice, or in other tissues. Fluorescence is present in the cortex and olfactory bulb only, however, demonstrating the cre-dependence of this reporter.
The donating laboratory achieved photoconversion (PC) of CaMPARI2 in vivo via implanted optic fibers (500um diameter, NA = 0.63). High-power LEDs were used as a source of 405 nm PC light. The power of the PC light at the tip of the light guide was up to 150mW/mm2, depending on the targeted depth. They indicate that the power should be optimized for each experiment. For exposure times in the range of minutes, PC light should be pulsed. They used 4 sec on/1 sec off over 30 min for 150 mW/mm2. Optic fibers and high-power LEDs can be purchased from companies such as Prizmatix. Additional materials needed for fiber connections are available from companies such as Precision Fiber Products.
A monoclonal antibody that specifically recognizes the photoconverted red form has been developed by Eric Schreiter at Janelia Research Campus, allowing immunohistochemical detection and selective amplification of the red CaMPARI2 signal in fixed cells or tissue.
CaMPARI (Calcium Modulated Photoactivatable Ratiometric Integrator) is a photoconvertible protein construct that enables imaging of the integrated calcium activity of large populations of cells over defined time windows. It is derived from the stony coral protein EOS, which irreversibly shifts its fluorescence. It photoconverts from green to red in the presence of violet light. By fusing calcium binding calmodulin and a calmodulin/Ca2+ binding peptide (M13) to either end, CaMPARI photoconversion becomes calcium dependent.
Calcium binding affinity variants of the original CaMPARI (CaMPARI1) flurorescent protein were identified (CaMPARI1_W391F-V398L_Q142V-F198Y-C202T-L217I-N345S) and incorporated in CaMPARI2 via targeted mutagenesis of amino acids around the fluorescent protein chromophore and at the protein interface between the fluorescent protein and the calcium-sensitive domains.
A CAG-loxP-Stop-loxP-CaMPARI2::attB-PGK-FRT-Neo-pA-attP vector was targeted to intron 1 of the Gt(ROSA)26Sor gene via homologous recombination in (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells. This line was backcrossed to C57BL/6J for 6 or 7 generations by the donating laboratory.
|Allele Name||targeted mutation 1, Mario R Capecchi|
|Allele Type||Targeted (Conditional ready (e.g. floxed), Reporter)|
|Allele Synonym(s)||CaMPARIlsl; CaMPARI2|
|Gene Symbol and Name||Gt(ROSA)26Sor, gene trap ROSA 26, Philippe Soriano|
|Strain of Origin||(129S6/SvEvTac x C57BL/6NCrl)F1|
|Molecular Note||The targeting construct contains a CAG promoter followed by a loxP-Stop-loxP-CaMPARI2::attB-PGK-FRT-Neo-pA-attP integrated into intron 1 of the Gt(ROSA)26Sor locu. CaMPARI (Calcium Modulated Photoactivatable Ratiometric Integrator) is a photoconvertible protein construct derived from the stony coral protein Eos. CaMPARI and irreversibly photoconverts from green to red in the presence of violet light and high intracellular calcium. The CaMPARI protein construct includes a calcium binding calmodulin with a calmodulin/Ca<2+> binding peptide (M13) fused to either end of the protein making the photoconversion calcium dependent. Calcium binding affinity variants of the original CaMPARI (CaMPARI1) fluorescent protein were identified (CaMPARI1_W391F-V398L_Q142V-F198Y-C202T-L217I-N345S) and incorporated in CaMPARI2 via targeted mutagenesis of amino acids around the fluorescent protein chromophore and at the protein interface between the fluorescent protein and the calcium-sensitive domains.|
Heterozygotes and homozygotes are viable and fertile, demonstrate normal development and weight gain, and show no abnormal behaviors.
When using the R26-CAG-lsl-CaMPARI2 , CaMPARIlsl
mouse strain in a publication, please cite the originating article(s) and include JAX stock #034240 in your Materials and Methods section.
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