B6;129S6-Igs7^{tm1(tetO-QuasAr2/GCaMP6f)Acoh}/J

Stock number: 029678
Product name: CaViAr Ai142
Research areas: Neurobiology Research, Research Tools

Description

Expression of QuasAr2-GCaMP6F (CaViAr; an optogenetic construct which enables conditional voltage and calcium imaging) is under the control of a tetracycline response element (TRE; tetO) and a floxed-STOP integrated in the Igs7 (TIGRE) locus. Functionality of CaViAr requires the expression of tet-activator (tTA or rtTA+dox) as well as Cre recombinase-mediated excision of the floxed-STOP. This is line Ai142.

Detailed description

Ai142 is a Cre recombinase-dependent and tetracycline-controlled transactivator (tTA)-dependent mouse line that conditionally expresses QuasAr2-GCaMP6f (CaViAr), an optogenetic construct which enables conditional voltage and calcium imaging. The animals express a near infrared Archaerhodopsin-derived voltage indicator fused to a green fluorescent calcium indicator (QuasAr2-TS-GCaMP6f) via a targeted tetracycline responsive element (TRE; tetO) and floxed-STOP knock-in of the Igs7 (intergenic site 7; also called TIGRE) locus. Both tet-activator (tTA or rtTA+dox) and Cre recombinase expression are required to achieve CaViAr expression.

The QuasAr2 voltage indicator is optimally excited with a wavelength between 630–640 nm, and emits in a broad band from 660-760 nm. The fluorescence quantum yield of QuasAr2 is low (~1%), so excitation must be provided via a high intensity laser (>100 W/cm²) and detection must be through a high numerical aperture objective and onto a highly sensitive camera (e.g. scientific CMOS or EMCCD). To detect single action potentials requires imaging at >500 frames/s. QuasAr2 reports membrane voltage with a sensitivity of ΔF/F ~90%/100 mV and a response time of ~1.5 ms.

Good expression and function are seen in Camk2a-expressing neurons when the Ai142 mice are crossed with Camk2a-tTA (see Stock No. 003010) and Camk2a-Cre animals.

The Ai142 construct is safe to express throughout the whole animal.

Development

The following were introduced, in order, to the Igs7 (TIGRE) locus via homologous recombination in (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells: tetracycline response element (TRE; tetO)-loxP-Stop-loxP-CaViAr. CaViAr is composed of QuasAr2, a single golgi trafficking signal (TS), GCaMP6f, and woodchuck hepatitis virus posttranscriptional regulatory element (WPRE).

The intersectional expression strategy is shown in Fig. 2a of Madisen et al., 2015 Neuron 85:942 PMID:25741722.

Details of the engineering and characterization of the Optopatch2 construct are in Hochbaum et al., 2014 Nat Methods 11:825 PMID:24952910.

Addition of a golgi trafficking sequence (TS; a C-terminal TLM mutation) improved targeting to the plasma membrane. Details of the engineering and characterization of the GCaMP6f construct are in Chen et al., 2013 Nature 499:295 PMID:23868258.

Resultant chimeric mice (founder line Ai142) were backcrossed to C57BL/6NCrl for 6-7 generations by the donating lab.

A 48 SNP (single nucleotide polymorphism) panel analysis, with 43 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. Three of the 27 markers throughout the genome were segregating, and 1 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 had an incomplete backcross.

Allele

Symbol: Igs7^{tm1(tetO-QuasAr2/GCaMP6f)Acoh}
Name: targeted mutation 1, Adam Cohen
MGI accession ID: MGI:5796834
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inducible
Synonyms: Ai142
Marker symbol: Igs7
Marker name: intergenic site 7
Marker synonyms: Iis7; TIGRE
Chromosome: 9
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: GFP,Green Fluorescent Protein,
Site of expression: CaViAr (QuasAr2/GCaMP6f) expression is achieved in cre expressing tissues after tetracycline-controlled transactivator (tTA) and cre recombinase expression.
Molecular note: The targeting construct contains, in order, a tetracycline response element (TRE; tetO)-loxP-Stop-loxP-CaViAr. CaViAr is composed of QuasAr2, a single golgi trafficking signal (TS), GCaMP6f, and woodchuck hepatitis virus posttranscriptional regulatory element (WPRE).