B6;129S-Gt(ROSA)26Sor^{tm38(CAG-GCaMP3)Hze}/J

Stock number: 014538
Product name: Ai38 or Ai38(RCL-GCaMP3)
Research areas: Cell Biology Research, Neurobiology Research, Research Tools

Description

A C57BL/6J congenic version of this Ai38 strain will remain available as Stock No. 029043).

Ai38 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

Ai38 mice heterozygous for the Rosa-CAG-LSL-GCaMP3-WPRE conditional allele are viable and fertile. A loxP-flanked STOP cassette prevents transcription of the downstream GCaMP3 fusion gene (see below for detailed description of GCaMP3). 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 Ai38 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. In the absence of calcium binding, low EGFP fluorescence is reported. Following calcium binding (such as neuronal activation) bright EGFP fluorescence is observed. GCaMP3 expression in tissues other than brain has not yet been evaluated by the donating investigator (April 2011). The phenotype of homozygous mice has not been characterized by the donating investigator.

Of note, the FRT sites flanking the mutation allow for additional targeted replacement of the reporter sequences through Flp-mediated recombination if so desired. Similarly, the attB/attP-flanked selection cassette may be removed by introduction of the site-specific bacteriophage PhiC31 integrase if so desired.

For characterization information, see images at the Allen Institute for Brain Science website (Ai38 images).

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.

Development

The Rosa-CAG-LSL-GCaMP3-WPRE targeting vector was designed by Dr. Hongkui Zeng (Allen Institute for Brain Science) to have (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), an FRT site, a loxP-flanked STOP cassette (with stop codons in all 3 reading frames and a triple polyA signal), 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 attB/attP-flanked PGK-FRT-Neo-polyA 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).

The entire Rosa-CAG-LSL-GCaMP3-WPRE targeting vector was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus via electroporation of (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells. Correctly targeted ES cells (clone Ai38) were selected.

Chimeric mice were bred with C57BL/6 wildtype mice and/or PhiC31-expressing mice (C57BL/6J congenic background; see Stock No. 007743); but some mice retaining the attB/attP-flanked PGK-frt-Neo-polyA cassette were propagated (see more details below).

The donating investigator reported that these mutant mice were then bred with C57BL/6J wildtype mice (but see SNP note below) for approximately four additional generations prior to sending to The Jackson Laboratory Repository. Upon arrival, sperm from Ai38 males was used to fertilize oocytes from C57BL/6J inbred mice (Stock No. 000664) in order to rederive the living colony at The Jackson Laboratory Repository.

All of the mice originally sent to The Jackson Laboratory Repository, as well as all the first generation of rederived offspring genotyped as completely co-segregating for the neo cassette and GCaMP3 portion of the Ai38 construct. As such, these mice harbor the Ai38 mutation (retaining the attB/attP-flanked PGK-frt-Neo-polyA cassette) rather than the Ai38Δneo mutation.

A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. While the 27 markers throughout the genome suggested a C57BL/6 genetic background, 3 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6J ; C57BL/6N genetic background.

Allele

Symbol: Gt(ROSA)26Sor^{tm38(CAG-GCaMP3)Hze}
Name: targeted mutation 38, Hongkui Zeng
MGI accession ID: MGI:5014745
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Chromosome: 6
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: GFP,Green Fluorescent Protein,
Site of expression: Cre excision of the stop signal results in expression of a circularly permutated EGFP in cre-expressing tissues.
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), an FRT site, a loxP-flanked STOP cassette (with stop codons in all 3 reading frames and a triple polyA signal), 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 attB/attP-flanked PGK-FRT-Neo-polyA 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).