B6.Cg-Igs7^{tm136.1(tetO-COP3*/COP4*/EYFP)Hze}/J

Stock number: 030216
Product name: Ai136(TITL-ReaChR-YFP)-D (or Ai136D)
Research areas: Neurobiology Research, Research Tools

Description

Ai136(TITL-ReaChR-YFP)-D (also called Ai136D) mice are a Cre/Tet-dependent, optogenetic line, created by targeted insertion at the Igs7 locus (TIGRE; an intergenic region on mouse chromosome 9 that allows reporter expression to be tightly regulated). Following Cre-mediated removal of the STOP cassette, they may be used to generate Tet-Off/Tet-On mutant animals with conditional (inducible/reversible) expression of the channelrhodopsin/EYFP fusion protein (ReaChR/EYFP). Subsequent illumination of ReaChR-expressing (EYFP fluorescent) cells with orange-to-red light leads to reversible photostimulation of action potential firing/neural activity in these cells.

Detailed description

Ai136(TITL-ReaChR-YFP)-D (also called Ai136D) mice are a Cre/Tet-dependent, optogenetic line, created by targeted insertion at the Igs7 locus (TIGRE; an intergenic region on mouse chromosome 9 that allows reporter expression to be tightly regulated). Ai136D mice harbor the TIGRE-Ins-TRE-LSL-ReaChR/EYFP conditional allele, designed with a modified Tet response element (TRE or tetO) and loxP-flanked STOP cassette upstream of an improved ReaChR/EYFP fusion protein (see detailed description below). When bred with other mice expressing Cre recombinase, tetracycline-controlled transactivator protein (tTA) and/or reverse tetracycline-controlled transactivator protein (rtTA), ReaChR expression in cells/tissues where the expression patterns of the individual promoters driving Cre and tTA/rtTA overlap can be regulated with tetracycline or its analog doxycycline (dox). Following induction of ReaChR expression (EYFP fluorescence), illuminating neurons with orange-to-red light (~590-630 nm) leads to rapid and reversible photostimulation of robust action potential firing activity in these cells.

Specifically, the donating investigator reports Ai136D mice have no reported levels of EYFP fluorescence in absence of Cre and tTA. When Ai136D mice are bred with tTA-driver and Cre-driver lines to create triple transgenic animals (ReaChR/EYFP+/Cre+/tTA+), cells expressing both tTA and Cre exhibit robust/bright fluorescence. Light-induced expression of the activation opsin occurs at levels sufficient to effectively depolarize/activate cortical neurons.

Native ReaChR-EYFP fluorescence has been observed in fixed and living brain sections in several Cre-defined cell types. 2P holographic activation of ReaChR-EYFP in in vitro brain slices from Rosa26-ztTA;Ntsr1-IRES-Cre;Ai136 mice elicits spiking. When using Ai136D with C57BL/6J-congenic driver lines Scnn1a-Tg3-Cre and ROSA26-ZtTA (see Stock Nos. 009613 or 012266, respectively), the donating investigator observes very efficient reporter function. ReaChR/EYFP expression/function in tissues other than brain has not yet been evaluated by the donating investigator (March 2017).

The donating investigator reports that heterozygous and homozygous mice are viable and fertile with no reported gross physical or behavioral abnormalities.

For characterization information, images may be available at the Allen Institute for Brain Science website (Ai136(TITL-ReaChR-YFP) images).

The bacterial opsins are retinal-binding proteins that combine a light-sensitive domain with an ion channel or pump; providing light-dependent ion transport, membrane potential alteration, and sensory functions to bacteria. The red-activatable channelrhodopsin chimera ReaChR is an orange-to-red light-driven (~590-630 nm) cation channel that depolarizes the cell and causes action potentials. As such, illuminating ReaChR-expressing cells with those wavelengths leads to rapid and reversible photostimulation of robust action potential firing activity in these cells. ReaChR is engineered with mammalian-codon optimization, improved membrane trafficking, higher photocurrents and faster kinetics compared with previous red-shifted channelrhodopsins (Lin et al. 2013 Nat Neurosci 16:1499). The ReaChR/EYFP fusion gene is composed of ReaChR fused in-frame at its C-terminus to an enhanced yellow fluorescent protein (EYFP) via a G-S-G linker.

Development

The Ai136(TITL-ReaChR-YFP)-D allele (or Ai136D) has a Tet response element, floxed-STOP cassette and ReaChR/EYFP fusion 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).

Specifically, the pTRE-LSL-ReaChR/EYFP replacement vector was designed with (from 5' to 3') an FRT3 site, two copies of chicken beta-globin HS4 insulator element (to reduce reporter gene expression in absence of transactivators), a modified Tet response element/promoter (TRE/tetO), a floxed-STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), the ReaChR/EYFP sequence (details below), a WPRE (to enhance mRNA stability), a BGH polyA, two copies of chicken beta-globin HS4 insulator element, an AttB site, a PGK-5'hygro cassette, an RNA splice donor and a FRT5 site.
(129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem cells previously targeted with FRT3::AttB::PGK-neoR-polyA::FRT5::splice acceptor::3'hygro cassette::SV40 polyA:AttP in TIGRE, were re-transfected with the pTRE-LSL-ReaChR/EYFP replacement vector and Flp recombinase vector for recombinase-mediated cassette exchange (RCME). Chimeric mice were bred with C57BL/6J-congenic PhiC31 mice (see Stock No. 007743) to remove the AttB/AttP-flanked PGK-hygro-SV40polyA cassette and replace it with the recombined AttB/AttP site (AttL). The Ai136D allele is therefore TIGRE-frt3-Ins-TRE-LSL-ReaChR/EYFP-WPRE-bGHpA-Ins-AttL. The resulting Ai136D mice were bred with C57BL/6J for several generations (and the PhiC31 gene was removed). In 2017, 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).

The ReaChR/EYFP fusion protein has the red-activatable channelrhodopsin variant ReaChR fused in-frame at its C-terminus (via a G-S-G linker) to an enhanced yellow fluorescent protein (EYFP). ReaChR, designed by Dr. Roger Tsien (UCSD), is a chimeric channelrhodopsin composed of transmembrane domains A-E and G of VChR1 (Volvox carteri channelrhodopsin 1 [cop3]), transmembrane domain F of VChR2 (V. carteri channelrhodopsin 2 [cop4]) and the ChEF/ChIEF variant N-terminal extracellular sequences (derived from Chlamydomonas reinhardtii channelrhodopsin 1 [cop3]). The ChEF/ChIEF, VChR1 and VChR2 sequences are mammalian-codon optimized, and transmembrane domain C harbors two point mutations encoding I170V (to accelerate rate of channel closure while retaining reduced inactivation) and L171I (to enhance activation by red light [~590 nm]). The ChEF/ChIEF sequences were used to impart its superior membrane trafficking. [Lin et al. 2013 Nat Neurosci 16:1499]

The TRE promoter used here is Tet-responsive P_tight; containing a modified Tet response element (TRE_mod) composed of seven direct 36 bp repeats (each containing the 19 bp tet operator sequence [tetO]). TRE_mod is just upstream of a minimal human cytomegalovirus promoter (P_minCMVΔ), which lacks the enhancer that is part of the complete CMV promoter. Consequently, P_tight is silent in the absence of tTA or rtTA binding to tetO.

Allele

Symbol: Igs7^{tm136.1(tetO-COP3*/COP4*/EYFP)Hze}
Name: targeted mutation 136.1, Hongkui Zeng
MGI accession ID: MGI:5903984
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inducible
Synonyms: Ai136(TITL-ReaChR-YFP)-D; Ai136D
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: YFP,Yellow Fluorescent Protein,jellyfish
Site of expression: When bred with other mice expressing Cre recombinase, tetracycline-controlled transactivator protein (tTA) and/or reverse tetracycline-controlled transactivator protein (rtTA), ReaChR expression in cells/tissues where the expression patterns of the individual promoters driving Cre and tTA/rtTA overlap can be regulated with tetracycline or its analog doxycycline (dox).
Molecular note: The replacement vector was designed with (from 5' to 3') an FRT3 site, two copies of chicken beta-globin HS4 insulator element, a modified Tet response element/promoter (TRE/tetO), a floxed-STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), the ReaChR/EYFP sequence (details below), a WPRE, a BGH polyA, two copies of chicken beta-globin HS4 insulator element, an AttB site, a PGK-5'hygro cassette, an RNA splice donor and a FRT5 site. Embryonic stem cells previously targeted with FRT3::AttB::PGK-neoR-polyA::FRT5::splice acceptor::3'hygro cassette::SV40 polyA:AttP in TIGRE, were re-transfected with the pTRE-LSL-ReaChR/EYFP replacement vector and Flp recombinase vector for recombinase-mediated cassette exchange (RCME).PhiC31-mediated recombination removed the AttB/AttP-flanked sequence (PGK-Neo-polyA::frt5::RNA splice acceptor::3'hygro-polyA) and replaced it with the recombined AttB/AttP site (AttL). The ReaChR/EYFP fusion protein has the red-activatable channelrhodopsin variant ReaChR fused in-frame at its C-terminus (via a G-S-G linker) to an enhanced yellow fluorescent protein (EYFP). ReaChR is a chimeric channelrhodopsin composed of transmembrane domains A-E and G of VChR1 (Volvox carteriï¿œchannelrhodopsin 1 [cop3]), transmembrane domain F of VChR2 (V. carteriï¿œchannelrhodopsin 2 [cop4]) and the ChEF/ChIEF variant N-terminal extracellular sequences (derived fromï¿œChlamydomonas reinhardtiiï¿œchannelrhodopsin 1 [cop3]). The ChEF/ChIEF, VChR1 and VChR2 sequences are mammalian-codon optimized, and transmembrane domain C harbors two point mutations encoding I170V (to accelerate rate of channel closure while retaining reduced inactivation) and L171I (to enhance activation by red light [~590 nm]).