B6;129S6-Igs7^{tm4(tetO-Rpl10*/GCaMP8s,CAG-tTA2)Genie}/J

Stock number: 039267
Product name: TIGRE2-RiboL1-jGCaMP8s-IRES-tTA2
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

TIGRE2-RiboL1-jGCaMP8s-IRES-tTA2 knock-in mice co-express a fusion of jGCaMP8s genetically encoded calcium indicator (GECI) tethered to a component of the large ribosomal subunit (Rpl10) via an optimized linker sequence (L1) and a tetracycline-controlled transactivator (tTA2) in a Cre recombinase-dependent manner from the intergenic Igs7 (TIGRE) locus. By tethering jGCaMP8s to the ribosome, protein expression is restricted to the cell soma thereby reducing neuropil signals. The addition of an internal ribosome entry site (IRES) introduced upstream of tTA and the omission of a WPRE enhancer element downstream ameliorate previously reported tTA overexpression toxicity issues. jGCaMP8s is a fast rise, medium decay green fluorescent-based GECI with improved sensitivity and faster kinetics.

Detailed description

These TIGRE2-RiboL1-jGCaMP8s-IRES-tTA2 mice (described on the Janelia Research Campus website) are similar to STOCK Igs7^{tm2(tetO-GCaMP8s,CAG-tTA2)Genie}/J animals (Stock No. 037719). They co-express a fusion of jGCaMP8s genetically encoded calcium indicator (GECI) tethered to a component of the large ribosomal subunit (Rpl10) via an optimized linker sequence (L1) and a tetracycline-controlled transactivator (tTA2) in a Cre recombinase-dependent manner from the intergenic Igs7 (TIGRE) locus. By tethering jGCaMP8s to the ribosome, protein expression is restricted to the cell soma thereby reducing neuropil signals.

The expression of this improved jGCaMP8s (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 jGCaMP8s indicator is a fast rise, medium decay green fluorescent-based GECI with improved sensitivity and faster kinetics in targeted neuronal subpopulations.

Expression of tTA is attenuated by the introduction of an IRES (internal ribosome entry site) upstream of tTA and the omission of a WPRE enhancer element downstream. These modifications ameliorate 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

These TIGRE2-RiboL1-jGCaMP8s-IRES-tTA2 mice are similar to STOCK Igs7^{tm2(tetO-GCaMP8s,CAG-tTA2)Genie}/J animals (Stock No. 037719), but carry an additional Ribo-tag sequence (RiboL1, modified from Rpl10, ribosomal protein L10).

An HS4 insulator-Tet response element/promoter (pTRE2)-loxP-Stop-loxP-RiboL1-jCaMP8s-WPRE-pA-HS4 insulator-pCAG-lox2272-Stop-lox2272-FLAG-IRES-tTA2-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.

jGCaMP8s incorporates a hexa-histidine affinity tag, an endothelial nitric oxide synthase peptide (ENOSP; modified with N19T, S24I, and S26M) 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 Q315H), and improved amino acid linkers. The overall jCaMP8s 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").

The targeted ES cells were aggregated with 8-cell CD1 outbred embryos. The strain was maintained on a mixed background including C57BL/6 and 129S6/SvEvTac by the donating laboratory prior to sending males to The Jackson Laboratory Repository. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Allele

Symbol: Igs7^{tm4(tetO-Rpl10*/GCaMP8s,CAG-tTA2)Genie}
Name: targeted mutation 4, Janelia GENIE Project Team
MGI accession ID: MGI:7607114
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inducible; Transactivator; Epitope tag
Synonyms: TIGRE2-RiboL1-jGCaMP8s-IRES-tTA2
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 an HS4 insulator-Tet response element/promoter (pTRE2)-loxP-Stop-loxP-RiboL1-CaMP8s-WPRE-pA-HS4 insulator-pCAG-lox2272-Stop-lox2272-FLAG-IRES-tTA2-pA cassette introduced to the Igs7 intergenic region. RiboL1 is a ribo-tag sequence modified from Rpl10, ribosomal protein L10. gCaMP8s (sensitive) incorporates a hexa-histidine affinity tag, an endothelial nitric oxide synthase peptide (ENOSP; modified with N19T, S24I, and S26M), a circularly permutated green fluorescent protein (cpGFP; modified with Q88E), the rat calcium-binding protein calmodulin (CaM; modified with F286Y and Q315H), 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)