B6N;129-Gt(ROSA)26Sor^{tm1(CAG-GCaMP3)Dbe}/J

Stock number: 028764
Product name: Rosa26-lsl-GCaMP3
Research areas: Neurobiology Research, Research Tools

Description

Rosa26-lsl-GCaMP3 mice express the fluorescent calcium indicator protein GCaMP3 after exposure to Cre recombinase. Following subsequent calcium binding (such as neuronal activation), bright EGFP fluorescence is observed.

Detailed description

Mice heterozygous for the Rosa26-lsl-GCaMP3 conditional allele have a loxP-flanked STOP cassette prevents transcription of the downstream GCaMP3 fusion gene. The donating investigator reports that the presence of the neomycin resistance cassette did not have any adverse effects on the expression of GCaMP3 or the health and survival of these mice. Because the CAG promoter driven reporter construct was targeted for insertion into the Gt(ROSA)26Sor locus, GCaMP3 expression is determined by which tissue(s) express Cre recombinase. When bred to mice that express Cre recombinase, the resulting offspring will have the STOP cassette deleted in the cre-expressing tissues; resulting in expression of the fluorescent calcium indicator protein, GCaMP3. The donating investigator reports that mice do not express GCaMP3 prior to introduction of Cre recombinase. Following exposure to Cre recombinase, GCaMP3 expression (EGFP fluorescence) is detected in the cre-expressing tissues. Following calcium binding (such as neuronal activation) bright EGFP fluorescence is observed.

GCaMP3 is a fluorescent calcium indicator. The GCaMP3 fusion gene contains the chicken smooth muscle M13 fragment of myosin light chain kinase (MYLK), a circularly permutated EGFP sequence (with several amino acid substitutions designed to increase dynamic range and baseline fluorescence), and a rat calmodulin (Calm) sequence modified to increase the fluorescence change for small calcium transients. In the absence of calcium binding, dim EGFP fluorescence is expected as there is a pore from the outside of its barrel into the chromophore. Upon calcium binding, this pore becomes occupied and fluorescence is increased.

When bred to STOCK Tg(Slc1a3-cre/ERT)1Nat/J mice (Stock No. 012586), expressing tamoxifen-inducible Cre Recombinase in astrocytes of the brain, resulting mice express GCaMP3 in 35% of cortical astrocytes, and 100% of Gergmann glia cells following tamoxifen administration. In vivo two-photon imaging was used to define the activity patterns of cortical and cerebellar astrocytes during increased Ca²⁺ release during locomotion.

Development

The Rosa26-lsl-GCaMP3 targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), a loxP-flanked STOP cassette (3x SV40 polyA), a GCaMP3 fusion gene, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a BGH polyA signal, and an frt-flanked neomycin resistance cassette in reverse orientation to the gene.

The GCaMP3 fusion gene contains a cDNA sequence encoding the chicken smooth muscle M13 fragment of myosin light chain kinase, a circularly permutated EGFP (amino acid residues 149-238 followed by amino acid residues 1-144 (including M153K, V163A, S175G, D180Y, T203V, A206K, and V93I mutations to increase dynamic range and baseline fluorescence), and a DNA fragment encoding amino acid residues 2-148 of the rat calmodulin (modified to harbor the N60D mutation to increase the fluorescence change for small calcium transients).

The entire Rosa26-lsl-GCaMP3 targeting vector was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus via electroporation of 129-derived embryonic stem (ES) cells. Correctly targeted ES cells were selected and injected into 129SV recipient blastocysts. Resulting chimeric male were bred with C57BL/6N wildtype females and offspring were maintained on a C57BL/6N genetic background. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6NJ oocytes (Stock No. 005304).

Allele

Symbol: Gt(ROSA)26Sor^{tm1(CAG-GCaMP3)Dbe}
Name: targeted mutation 1, Dwight E Bergles
MGI accession ID: MGI:5659933
Generation method: Targeted
Attributes: Reporter
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Chromosome: 6
Strain of origin: 129
Expressed genes: GCaMP,Genetically encoded calcium indicator,; GFP,Green Fluorescent Protein,
Site of expression: When bred to mice that express Cre recombinase, the resulting offspring will have the STOP cassette deleted in the cre-expressing tissues; resulting in expression of the fluorescent calcium indicator protein, GCaMP3.
Molecular note: The Rosa-CAG-LSL-GCaMP3-WPRE targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), a loxP-flanked STOP cassette, a GCaMP3 cDNA, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a BGH polyA signal, and an FRT-flanked Neo cassette. The GCaMP3 fusion gene contains a cDNA sequence encoding the chicken smooth muscle M13 fragment of myosin light chain kinase, a circularly permutated EGFP (amino acid residues 149-238 followed by amino acid residues 1-144 (including M153K, V163A, S175G, D180Y, T203V, A206K, and V93I mutations to increase dynamic range and baseline fluorescence), and a DNA fragment encoding amino acid residues 2-148 of the rat calmodulin (modified to harbor the N60D mutation to increase the fluorescence change for small calcium transients).