B6;129S6-Igs7^{tm3(tetO-Optopatch3,CAG-tTA)Acoh}/J

Stock number: 029679
Product name: Optopatch3 Ai155
Research areas: Neurobiology Research, Research Tools

Description

Ai155 Optopatch3 mice, an improved version of Optopatch2, are a sensitive, fast optogenetic tool for targeted, simultaneous optical perturbation and optical measurement of membrane voltage. Conditional expression is dependent on cre-mediated excision of a floxed Stop cassette.

Detailed description

Ai155 is a cre recombinase-dependent mouse line that conditionally expresses Optopatch3, an improved optogenetic construct which enables simultaneous perturbation and optical readout of membrane potential. The animals express a blue-shifted channelrhodopsin actuator (CheRiff) and a near infrared Archaerhodopsin-derived voltage indicator (QuasAr3-mCitrine) via a targeted tetracyline responsive element (TRE; tetO), floxed Stop, and tetracycline-controlled transactivator (tTA) knock-in of the Igs7 (intergenic site 7; also called TIGRE) locus. Cre recombinase expression is required to achieve Optopatch3 expression. Constitutive expression of tTA removes the requirement for intersectional expression of tTA.

Optopatch3 mice represent an improved version of Optopatch2 (as found in Stock Nos. 029677 and 029678) by virtue of an additional QuasAr point mutation and modifications to the trafficking sequences. QuasAr3's bright mCitrine replaces QuasAr2's dark mOrange fusion protein. The EGFP fusion to CheRiff found in Optopatch2 has been removed in Optopatch3. The mCitrine component indicates the location and expression of QuasAr3.

The CheRiff optogenetic actuator is optimally excited by light with a wavelength between 450–490 nm, reaching 50% of maximum photocurrent at an illumination intensity of 25 mW/cm² (488 nm). CheRiff has an opening time of 4.5 ms, twofold faster than that of ChR2 H134R and fourfold faster than that of ChIEF. CheRiff has a closing time of 16 ms, similar to that of ChIEF and 1.5-fold faster than ChR2 H134R. Details of the engineering and characterization of the CheRiff are in: Hochbaum, Daniel R., et al. "All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins." Nature Methods 11.8 (2014): 825-833 (PMID 24952910).

The QuasAr3 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 QuasAr3 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. QuasAr3 reports membrane voltage with a sensitivity of ΔF/F ~90%/100 mV and a response time of ~1.5 ms. Expression and membrane localization of QuasAr3 can be verified via fluorescence of the mCitrine fusion, but one must remember that blue light used to excite mCitrine fluorescence will also activate CheRiff.

Ai155 has been tested with Camk2a-cre, somatostatin (Sst)-cre, Rbp4-cre and Scn10a (Na_v1.8)-cre; all show the predicted expression patterns.

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: tetO (tetracycline response element; TRE)-loxP-Stop-loxP-Optopatch3-CAG-tTA (tetracycline-controlled transactivator).

Optopatch3 is a bicistronic construct that coexpresses QuasAr3-mCitrine (an enhanced archaerhodopsin-3 (Arch)-based voltage indicator) and CheRiff (a sensitive blue-shifted optogenetic actuator) upon cre-mediated excision of the floxed Stop cassette.

Optopatch3 is functionally equivalent to Optopatch2 described in: Hochbaum, Daniel R., et al. "All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins." Nature Methods 11.8 (2014): 825-833 (PMID 24952910).

Optopatch3 has improved in vivo trafficking of the QuasAr voltage indicator, compared to Optopatch2.

Resultant chimeric mice (founder line Ai155) were backcrossed to C57BL/6NCrl for 6-7 generations by the donating lab (see SNP note below).

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. Eight of the 43 markers throughout the genome were segregating, suggesting an incomplete backcross. Also, 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 were on a mixed C57BL/6J ; C57BL/6N genetic background.

Allele

Symbol: Igs7^{tm3(tetO-Optopatch3,CAG-tTA)Acoh}
Name: targeted mutation 3, Adam Cohen
MGI accession ID: MGI:5796839
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inducible
Marker symbol: Igs7
Marker name: intergenic site 7
Chromosome: 9
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: YFP,Yellow Fluorescent Protein,jellyfish
Site of expression: Optopatch3 functionality is achieved in cre expressing tissues after tetracycline-controlled transactivator (tTA) and cre recombinase expression.
Molecular note: The targeting construct contains a tetO (tetracycline response element; TRE)-loxP-Stop-loxP-Optopatch3-CAG-tTA (tetracycline-controlled transactivator). Optopatch3 is a bicistronic construct that coexpresses QuasAr3-mCitrine (an enhanced archaerhodopsin-3 (Arch)-based voltage indicator) and CheRiff (a sensitive blue-shifted optogenetic actuator)