B6.Cg-Igs7^{tm134.1(tetO-COP4*H134R/EYFP)Hze}/J

Stock number: 031334
Product name: Ai134(TITL-ChR2H134R-EYFP)-D , Ai134D , Ai134(TITL-ChR2-YFP)-D
Research areas: Neurobiology Research, Research Tools

Description

Ai134(TITL-ChR2H134R-EYFP)-D mice (also called Ai134D or Ai134(TITL-ChR2-YFP)-D) 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 an improved channelrhodopsin2/EYFP fusion protein (ChR2(H134R)-EYFP). Subsequent illumination of ChR2(H134R)-expressing (EYFP fluorescent) cells with blue light leads to reversible photostimulation of action potential firing/neural activity in these cells.

Detailed description

Ai134(TITL-ChR2H134R-EYFP)-D mice (also called Ai134D or Ai134(TITL-ChR2-YFP)-D) 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). Ai134D mice harbor the TIGRE-Ins-TRE-LSL-ChR2(H134R)/EYFP conditional allele, designed with a modified Tet response element (TRE or tetO) and loxP-flanked STOP cassette upstream of the ChR2(H134R)/EYFP fusion protein (see detailed description below). When bred with other mice expressing Cre recombinase and tetracycline-controlled transactivator protein (tTA) or reverse tetracycline-controlled transactivator protein (rtTA), ChR2(H134R) 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 ChR2(H134R) expression (EYFP fluorescence), illuminating neurons with blue light (~450-490 nm) leads to rapid and reversible photostimulation of robust action potential firing activity in these cells.

Specifically, the donating investigator reports Ai134D mice have no reported levels of EYFP fluorescence in absence of Cre and tTA. When bred with tTA-driver and Cre-driver lines to create triple transgenic animals (ChR2(H134R)/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. ChR2(H134R)/EYFP expression/function in tissues other than brain has not yet been evaluated (January 2018).
For example, combining Ai134 with the moderately-expressing tTA-driver line ROSA26-ZtTA (see Stock No. 012266) and the cortical layer 4 Cre driver line Scnn1a-Tg3-Cre (Stock No. 009613) generates triple transgenic Scnn1a-Tg3-Cre;ROSA26-ZtTA;Ai134 animals. Those mice show good ChR2(H134R) expression and measurable photo-responses. The same may be expected for Ai134D. Additionally, the donating investigator advises against using very strong tTA drivers (e.g., Camk2a-tTA) in these triple transgenics - suggesting that the resulting high ChR2(H134R) expression levels lead to fluorescent aggregates in cells and/or aberrant morphology of labeled cells.

Ai134D heterozygotes are viable and fertile with no reported gross physical or behavioral abnormalities. To date (January 2018), it has not been attempted to make this strain homozygous.

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 ChR2(H134R)-EYFP fusion protein is composed of a Chlamydomonas reinhardtii-derived channelrhodopsin-2 that harbors a gain-of-function H134R substitution fused in-frame with an enhanced yellow fluorescent protein. The ChR2(H134R) is designed to cause larger stationary photocurrents compared to ChR2. This ChR2(H134R) functions as a blue light-driven cation channel that depolarizes the cell and causes action potentials. As such, illuminating ChR2(H134R)-expressing cells with blue light (450-490 nm) leads to rapid and reversible photostimulation of action potential firing activity in these cells.

Development

The Ai134(TITL-ChR2H134R-EYFP)-D allele (also called Ai134D or Ai134(TITL-ChR2-YFP)-D) has a Tet response element, floxed-STOP cassette and ChR2(H134R)-EYFP fusion gene 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-ChR2(H134R)-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 Tet response element/promoter (TRE; described below), a floxed-STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), the ChR2(H134R)-EYFP fusion 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-ChR2(H134R)-EYFP fusion 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 Ai134D allele is therefore TIGRE-frt3-Ins-TRE-LSL-ChR2(H134R)/EYFP-WPRE-bGHpA-Ins-AttL.
The resulting Ai134D mice were bred with C57BL/6J for several generations (and the PhiC31 transgene was removed). In 2018, male mice from generation N10 (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).

To create the ChR2(H134R)-EYFP fusion gene, a cDNA sequence encoding the first 315 amino acids of channelrhodopsin-2 (derived from the green alga Chlamydomonas reinhardtii) was modified with a gain-of-function H134R substitution (CAC to CGC) designed to cause larger stationary photocurrents. This ChR2(H134R) sequence was fused in-frame to the amino terminus of an enhanced yellow fluorescent protein sequence, resulting in the final ChR2(H134R)-EYFP fusion protein sequence.

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^{tm134.1(tetO-COP4*H134R/EYFP)Hze}
Name: targeted mutation 134.1, Hongkui Zeng
MGI accession ID: MGI:6151056
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inducible
Synonyms: Ai134; Ai134(TITL-ChR2-YFP)
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: COP4,Channelrhodopsin,Chlamydomonas; YFP,Yellow Fluorescent Protein,jellyfish
Site of expression: When bred with other mice expressing Cre recombinase and tetracycline-controlled transactivator protein (tTA) or reverse tetracycline-controlled transactivator protein (rtTA), ChR2(H134R) 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 vector is 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 Tet response element/promoter, a floxed-STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), the ChR2(H134R)-EYFP fusion sequence, 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. PhiC31-mediated recombination removed the AttB/AttP-flanked sequence and replaced it with the recombined AttB/AttP site (AttL).