B6.Cg-Igs7^{tm203(tetO-sChroME*/GCaMP7s,CAG-mRuby3,-tTA2)Daigl}/J

Stock number: 037939
Product name: Ai203
Research areas: Cell Biology Research, Neurobiology Research, Research Tools

Description

Ai203 (also called Ai203(TITL-sChroME-Kv2.1-GC7s-WPRE-ICL-nls-mRuby3-IRES2-tTA2-WPRE)) is a Cre recombinase-dependent, Tet-controllable, optogenetic, calcium sensor and fluorescent tool allele created by targeted insertion at the Igs7 locus (TIGRE). Exposure to Cre recombinase results in expression of sChroME*/GCaMP7s, mRuby3, and tTA2 in those cells where cre is expressed.

Detailed description

Ai203 mice harbor the TITL-sChroME-Kv2.1-GC7s-WPRE-ICL-nls-mRuby3-IRES2-tTA2-WPRE conditional allele, designed with a tetO promoter, loxP-STOP-loxP cassette and sChroME*/GCaMP7s sequence, and then a CAG promoter, Lox2272-STOP-Lox2272 cassette, mRuby3, and IRES-tTA2-WPRE sequence. sChroME is a soma-targeted (ST) ultrafast and highly potent opsin with 3-5× larger photocurrents than opsins commonly used for multiphoton optogenetics. sChroME*/GCaMP7s is the result of a sChroME* coupled to the fluorescent reporter GCaMP7s (an ultrasensitive EGFP fluorescent calcium detector of neuronal activity with slow decay and response kinetics).

Prior to recombinase exposure, this allele is designed to have the two STOP cassettes preventing expression of the downstream genes (sChroME*/GCaMP7s, mRuby3, and tTA2). As of January 2023, Ai203 heterozygotes and homozygotes are viable and fertile with no reported gross physical or behavioral abnormalities. However in March 2026, the donating laboratory states that TIGRE knock-in alleles with long stretch of repetitive sequence and multiple transcription units could potentially lead to instability and unequal cross-over during meiosis. As such, the donating laboratory strongly recommends maintaining this strain as heterozygous.

When bred to mice expressing Cre recombinase, the offspring will have both STOP cassettes deleted in the cells where cre is expressed, resulting in sChroME*/GCaMP7s, mRuby3, and tTA2 expression. sChroME* is fused to a rat Kv2.1 potassium channel gene (Kcnb1) C-terminal sequence encoding a 65 amino acid sequence that functions to restrict protein localization to the soma and proximal dendrites.

Specifically, the donating investigator reports Ai203 exhibits no visible sChroME*/GCaMP7s or mRuby3 fluorescence in the absence of cre. Of note, the Ai203 allele was designed with an IRES upstream of the mRuby3 and tTA2 to reduce/eliminate leakiness in the absence of Cre recombinase (Graybuck et al. 2021 Neuron [PMID:33789083]).

The donating investigator reports when Ai203 mice are bred with Slc17a7-IRES2-Cre mice (Stock No. 037512) with cre expression in the cortex, olfactory bulb and anterior olfactory nuclei, mice exhibit robust native GCaMP7s expression in fixed brain tissue, with more widespread mRuby3 expression seen in the cortex. This may be due to selective silencing of the TREtight promoter relative to CAG which is more ubiquitous.

When Ai203 mice are bred with Sst-IRES-cre mice (Stock No. 018973), with cre expression in somatostatin-expressing neurons, or Vgat-IRES-Cre mice (similar to Stock No. 028862), with cre expression in inhibitory GABAergic neuron cell bodies, resulting mice exhibit widespread mRuby3 expression with lower levels of GCaMP7s expression.

Development

This mutant allele/strain was generated in collaboration between Dr. Tanya Daigle (Allen Institute for Brain Science) and Dr. Hillel Adesnik (University of California, Berkeley).

The Ai203 allele (or Ai203(TITL-sChroME-Kv2.1-GC7s-WPRE-ICL-nls-mRuby3-IRES2-tTA2-WPRE)) has a Tet response element (tetO), loxP-STOP-loxP cassette and a sChroME*/GCaMP7s-WPRE sequence that is followed by a CAG promoter, Lox2272-STOP-Lox2272 cassette, mRuby3, and IRES-tTA2-WPRE sequence - all inserted into the T1 TIGRE locus (an intergenic region on chromosome 9 [between AB124611 and Carm1] determined to have low basal transcriptional activity and allow high cre and/or Tet inducibility).

129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem cells were targeted with the replacement vector using recombinase-mediated cassette exchange (RMCE). The resulting Ai203 mice were bred with C57BL/6J for seven generations. In 2023, males from generation N7 (black coat color) were sent to The Jackson Laboratory. Upon arrival, males are used to cryopreserve sperm. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

The donating investigator reports that, at least once during backcrossing, a heterozygous female was bred to a C57BL/6J inbred male (thus the Y chromosome of the congenic strain is of C57BL/6J origin).

sChroME*/GCaMP7s is the result of a sChroME* coupled to the fluorescent reporter GCaMP7s (an ultrasensitive EGFP fluorescent calcium detector of neuronal activity with slow decay and response kinetics). sChroME is a soma-targeted (ST) ultrafast and highly potent opsin with 3-5x larger photocurrents than opsins commonly used for multiphoton optogenetics.

Of note, the Ai203 allele was designed with an IRES upstream of the tTA2 to reduce/eliminate leakiness in the absence of cre recombinase (Graybuck et al. 2021 Neuron [PMID:33789083]).

Allele

Symbol: Igs7^{tm203(tetO-sChroME*/GCaMP7s,CAG-mRuby3,-tTA2)Daigl}
Name: targeted mutation 203, Tanya Daigle
MGI accession ID: MGI:7482000
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inducible; Transactivator
Synonyms: Ai203; Ai203^{TITL-sChroME-Kv2.1-GC7s-WPRE-ICL-nls-mRuby3-IRES2-tTA2-WPRE}; Igs7^{tm203(tetO-sChroME*/GCaMP7s,CAG-mRuby3,-tTA2)Tasic}
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 targeting vector has a Tet response element (tetO), loxP-STOP-loxP cassette and a sChroME*/GCaMP7s-WPRE sequence that is followed by a CAG promoter, Lox2272-STOP-Lox2272 cassette, mRuby3, and IRES-tTA2-WPRE sequence - all inserted into the T1 TIGRE locus (an intergenic region on chromosome 9 [between AB124611 and Carm1] determined to have low basal transcriptional activity and allow high cre and/or Tet inducibility). sChroME*/GCaMP7s is the result of a sChroME* coupled to the fluorescent reporter GCaMP7s (an ultrasensitive EGFP fluorescent calcium detector of neuronal activity with slow decay and response kinetics). sChroME is a soma-targeted (ST) ultrafast and highly potent opsin with 3-5x larger photocurrents than opsins commonly used for multiphoton optogenetics. Of note, the Ai203 allele was designed with an IRES upstream of the tTA2 to reduce/eliminate leakiness in the absence of cre recombinase.