B6;129S6-Polr2a^{Tn(pb-CAG-GCaMP5g,-tdTomato)Tvrd}/J

Stock number: 024477
Product name: PC-G5-tdT
Research areas: Neurobiology Research, Research Tools

Description

PC::G5-tdT mice express the fluorescent calcium indicator protein GCaMP5G variant and tdTomato red fluorescent protein in a Cre recombinase inducible system. Increases in intracellular calcium (such as through neuronal activation) leads to bright GCaMP5G fluorescence. PC::G5-tdT is suitable for use in applications related to functional imaging of activity in neurons, astrocytes and microglia.

Detailed description

These mice express Cre recombinase dependent genetically encoded calcium indicator variant, GCaMP5G, and the red fluorescent protein tdTomato, under the control of the of the CAG promoter targeted in an intergenic region downstream of the endogenous Polr2a gene. GCaMP consists of a circularly permutated green fluorescent protein (cpGFP), the calcium-binding protein calmodulin (CaM), and CaM-interacting M13 peptide. The GCaMP5G variant was engineered from GCaMP3 and is an improved version with higher signal-to-noise ratio and higher ΔF/F_o compared to the GCaMP3 variant. When crossed to a strain expressing Cre recombinase to remove floxed STOP cassette, the resulting progeny mice will express both GCaMP5G and tdTomato in the cre-expressing tissues. tdTomato was included to assist in identification of GCaMP5G-expressing cells. However, FLP recombinase can be used to excise the FRT-flanked IRES-tdTomato, if desired. Mice that are homozygous for the targeted mutation are viable and fertile.

When crossed to a global Cre-deleter line (see Stock No. 004302 for example), GCaMP5G and tdTomato expression in was detected at high levels in the central nervous system throughout development and at lower levels in visceral organs.

Development

A targeting vector containing a floxed STOP cassette followed by a NEO selection cassette in opposite orientation, the genetically encoded calcium indicator GCaMP5G, a FRT flanked IRES-tdTomato, and woodchuck hepatitis posttranscriptional regulatory element (WPRE), under the control of the CAG promoter, was utilized in the construction of this reporter. This construct was flanked by piggyBAC transposase inverted terminal repeats and the entire reporter cassette was inserted approximately 1 kb 3' of the last exon of the Polr2a gene. The construct was electroporated into (129S6/SvEvTac x C57BL/6NCr)F1 derived G4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6 blastocysts. The resulting chimeric animals were crossed to C57BL/6 mice. Heterozygotes were crossed to generate homozygotes. Upon arrival at The Jackson Laboratory, the mice were crossed to C57BL/6J (Stock No. 000664) at least once to establish the colony.

Allele

Symbol: Polr2a^{Tn(pb-CAG-GCaMP5g,-tdTomato)Tvrd}
Name: transposon insertion 1, Petr Tvrdik
MGI accession ID: MGI:5560335
Generation method: Transposon induced
Attributes: Conditional ready (e.g. floxed); Reporter; No functional change
Marker symbol: Polr2a
Marker name: polymerase (RNA) II (DNA directed) polypeptide A
Chromosome: 11
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: GCaMP,Genetically encoded calcium indicator,; RFP,Red Fluorescent Protein,coral
Site of expression: After breeding mice carrying this allele with a strain expressing Cre recombinase to remove the floxed STOP cassette, resulting progeny will express both GCaMP5G and tdTomato in the cre-expressing tissues.
Molecular note: The transposon contains a floxed STOP cassette followed by a NEO selection cassette in opposite orientation, the genetically encoded calcium indicator GCaMP5g, a FRT-flanked IRES-tdTomato, and woodchuck hepatitis posttranscriptional regulatory element (WPRE), under the control of the CAG promoter. This construct was flanked by piggyBAC transposase inverted terminal repeats and the entire reporter cassette was inserted approximately 1 kb 3' of the last exon of the Polr2a gene.