STOCK Igs7^{tm1(tetO-GCaMP8m,CAG-tTA2)Genie}/J

Stock number: 037718
Product name: TIGRE2‐jGCaMP8m‐IRES‐tTA2‐WPRE
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

TIGRE2-jGCaMP8m-IRES-tTA2-WPRE knock-in mice co-express the jGCaMP8m genetically encoded calcium indicator (GECI) and a tetracycline controlled transactivator (tTA2) in a Cre recombinase-dependent manner from the intergenic Igs7 (TIGRE) locus. An internal ribosome entry site (IRES) introduced upstream of tTA ameliorates previously reported tTA overexpression toxicity issues. jGCaMP8m is noted for its faster decay rate and offers a useful compromise between sensitivity and kinetics.

Detailed description

The design of these TIGRE2-jGCaMP8m-IRES-tTA2-WPRE mice was based on that of the Ai162 strain (PMID: 30007418; similar to Stock No. 031562).

The expression of this improved jGCaMP8m (a genetically encoded calcium indicator; GECI) and a tetracycline controlled tetracycline controlled transactivator (tTA2) is Cre recombinase‐dependent and under the regulation of the tetracycline‐responsive promoter element (TRE; tetO) that have been introduced to the intergenic Igs7 locus (also called TIGRE). The jGCaMP8m indicator, offering improved sensitivity and faster kinetics, is stably expressed in targeted neuronal subpopulations. It is noted for its faster decay rate and offers a useful compromise between sensitivity and kinetics.

Expression of tTA is attenuated by the introduction of an IRES (internal ribosome entry site) upstream of tTA. This modification ameliorates previously reported issues with overexpression of tTA. Offspring from pan‐excitatory and pan‐inhibitory crosses displayed no perinatal lethality, epileptiform activity, or signs of neurodegeneration.

These mice were made by the Genetically-Encoded Neuronal Indicator and Effector (GENIE) Project at the HHMI's Janelia Farm Research Campus.

Development

An HS4 insulator-Tet response element/promoter (pTRE2)-loxP-Stop-loxP-jCaMP8m-WPRE-pA-HS4 insulator-pCAG-lox2272-Stop-lox2272-IRES-tTA2-WPRE-pA cassette was introduced to the Igs7 (TIGRE) intergenic region on mouse Chromosome 9 through FLP recombinase-mediated cassette exchange in Ai99 Flp-in embryonic stem (ES) cells. The cell line, carrying a docking site in the Igs7 (TIGRE) intergenic region, is derived from G4 ES cells on a (129S6/SvEvTac x C57BL/6)F1 genetic background.

jGCaMP8m incorporates a hexa-histidine affinity tag, an endothelial nitric oxide synthase peptide (ENOSP; modified with N19T, S24I, A25G, and S26R) that replaces the RS20 (previously referred to as M13) peptide used in previous GCaMP strains, a circularly permutated green fluorescent protein (cpGFP; modified with Q88E), the rat calcium-binding protein calmodulin (CaM; modified with F286Y), and improved amino acid linkers. The overall jCaMP8m construct is: Met-His6 tag-peptide-linker 1-cpGFP-linker 2-CaM. These mice were made by the Genetically-Encoded Neuronal Indicator and Effector (GENIE) Project at the HHMI's Janelia Research Campus (indicated by the "j"). More information on the construct may be found in Nature 615, 884–891 (2023), PMID: 36922596.

The targeted ES cells were aggregated with 8-cell CD1 outbred embryos. The strain was maintained on a mixed background including CD1, C57BL/6, and 129S6/SvEvTac by the donating laboratory.

Upon arrival at The Jackson Laboratory, sperm was cryopreserved. Cryorecovery/live colony development will involve C57BL/6J (Stock No. 000664).

Allele

Symbol: Igs7^{tm1(tetO-GCaMP8m,CAG-tTA2)Genie}
Name: targeted mutation 1, Janelia GENIE Project Team
MGI accession ID: MGI:7410949
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inducible; Transactivator; Epitope tag
Synonyms: TIGRE2-jGCaMP2m-IRES-tTA-WPRE
Marker symbol: Igs7
Marker name: intergenic site 7
Marker synonyms: Iis7; TIGRE
Chromosome: 9
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Molecular note: The transgenic construct contains HS4 insulator-Tet response element/promoter (pTRE2)-loxP-Stop-loxP-CaMP8m-WPRE-pA-HS4 insulator-pCAG-lox2272-Stop-lox2272-FLAG-IRES-tTA2-WPRE-pA cassette was introduced to the Igs7 intergenic region. GCAMP8m (medium) incorporates a hexa-histidine affinity tag, an endothelial nitric oxide synthase peptide (ENOSP; modified with N19T, S24I, A25G, and S26R), a circularly permutated green fluorescent protein (cpGFP; modified with Q88E), the rat calcium-binding protein calmodulin (CaM; modified with F286Y), and LKI amino acid linkers that replace the RS20 (previously referred to as M13) linkers.
General note: ES cell = made from Ai99 Flp-in mouse (derived from G4)