B6N;129-Gt(ROSA)26Sor^{tm2(CAG-mGCaMP3)Dbe}/J

Stock number: 030170
Product name: Rosa26-lsl-mGCaMP3
Research areas: Neurobiology Research, Research Tools

Description

Rosa26-lsl-mGCaMP3 mice express the membrane-associated 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-mGCaMP3 conditional allele have a loxP-flanked STOP cassette that prevents transcription of the downstream membrane-associated mGCaMP3 fusion gene. The donating investigator reports that the presence of the neomycin resistance cassette does 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, mGCaMP3. The donating investigator reports that mice do not express mGCaMP3 prior to introduction of Cre recombinase. Following exposure to Cre recombinase, mGCaMP3 expression (EGFP fluorescence) is detected on the cell membrane of cells in 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. To localize GCaMP3 to the plasma membrane, a gene sequence encoding the first 8 amino acids of the modified Marcks (myristoylated alanine rich protein kinase C substrate) sequence (MGCCFSKT) was fused to the transcription initiation codon.

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 cortical astrocyte microdomains following tamoxifen administration. In vivo two-photon imaging was used to define the activity patterns of cortical and cerebellar astrocytes during increased spontaneous Ca²⁺ release as seen in CNS injury and Alzheimer’s disease.

Development

The Rosa26-lsl-mGCaMP3 targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), a loxP site, a frt-flanked neomycin resistance cassette in reverse orientation to the gene, a STOP cassette (3x SV40 polyA), a second loxP site, a GCaMP3 fusion gene, and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability) followed by a BGH polyA signal.

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). To localize GCaMP3 to the plasma membrane, a gene sequence encoding the first 8 amino acids of the modified Marcks (myristoylated alanine rich protein kinase C substrate) sequence (MGCCFSKT) was fused to the transcription initiation codon.

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 males 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^{tm2(CAG-mGCaMP3)Dbe}
Name: targeted mutation 2, Dwight E Bergles
MGI accession ID: MGI:6111492
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Synonyms: Rosa26-lsl-mGCaMP3
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: 129
Expressed genes: GCaMP,Genetically encoded calcium indicator,; GFP,Green Fluorescent Protein,
Site of expression: Following exposure to Cre recombinase, mGCaMP3 expression (EGFP fluorescence) is detected on the cell membrane of cells in cre-expressing tissues.
Molecular note: The targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), a loxP site, an FRT-flanked neomycin resistance cassette in reverse orientation to the gene, a STOP cassette (3x SV40 polyA), a second loxP site, a GCaMP3 fusion gene, and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability) followed by a BGH polyA signal. 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). To localize GCaMP3 to the plasma membrane, a gene sequence encoding the first 8 amino acids of the modified Marcks (myristoylated alanine rich protein kinase C substrate) sequence (MGCCFSKT) was fused to the transcription initiation codon. The targeting vector is inserted between exons 1 and 2.