Overview

Also Known As:Ai90(TITL-Chronos)-D or Ai90D

Ai90(TITL-Chronos)-D (also called Ai90D) 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/EGFP fusion protein (Chronos/EGFP). Subsequent illumination of Chronos-expressing (EGFP fluorescent) cells with blue-to-green light leads to reversible photostimulation of action potential firing/neural activity in these cells.

Donating Investigator

Hongkui Zeng, Allen Institute for Brain Science

Genetic overview

Genetic Background	Generation

Igs7^{tm90.1(tetO-COP4/EGFP)Hze}*

Allele Type	Gene Symbol	Gene Name
Targeted (Conditional ready (e.g. floxed), Reporter, Inducible)	Igs7	intergenic site 7

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Research Applications

Research Tools
Neurobiology Research

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Base Price

Starting at:

$2,854.50 Domestic price Cryo Recovery

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Details

Detailed Description

Ai90(TITL-Chronos)-D 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). Ai90D mice harbor the TIGRE-Ins-TRE-LSL-Chronos conditional allele, designed with a modified Tet response element (TRE or tetO) and loxP-flanked STOP cassette upstream of an improved Chronos/EGFP 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), Chronos 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 Chronos expression (EGFP fluorescence), illuminating neurons with blue-to-green light leads to reversible photostimulation of action potential firing/neural activity in these cells.

Specifically, the donating investigator reports Ai90D mice have no reported levels of EGFP fluorescence in absence of Cre and tTA. When Ai90D mice are bred with tTA-driver and Cre-driver lines to create triple transgenic animals (Chronos+/tTA+/Cre+), cells expressing both tTA and Cre exhibit robust fluorescence. Light-induced expression of the activation opsin is expected to occur at levels sufficient to effectively depolarize/activate cortical neurons. Chronos expression/function in tissues other than brain has not yet been evaluated by the donating investigator (March 2014).

Heterozygous Ai90D mice are viable and fertile with no reported gross physical or behavioral abnormalities. The donating investigator has not attempted to generate homozygous Ai90D mice to date (March 2014).

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 Stigeoclonium helveticum-derived channelrhodopsin ShChR (Chronos) is a blue-to-green light-driven cation channel that depolarizes the cell and causes action potentials. As such, illuminating Chronos-expressing cells with blue-to-green light (~470-530 nm) leads to rapid and reversible photostimulation of robust action potential firing activity in these cells. Chronos has high light sensitivity and faster kinetics than previous channelrhodopsins. The action spectrum of Chronos is shifted to longer wavelengths compared to ChR2. In addition, Chronos is especially well suited to be paired with a red-shifted channelrhodopsin in order to allow for two light color activation of independent populations of neurons. The Chronos/EGFP fusion gene is composed of a mammalian codon-optimized Chronos fused in-fame to the N-terminus of EGFP.

Development

The Ai90(TITL-Chronos)-D allele (also called Ai90Δhygro or Ai90D) has a Tet response element, floxed-STOP cassette and the Chronos/EGFP fusion protein 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-Chronos/EGFP 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 (TRE or tetO), a floxed-STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), the channelrhodopsin/EGFP fusion protein coding sequence (Chronos/EGFP; details below), a WPRE sequence (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-Chronos/EGFP 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-hygromycin-SV40polyA cassette and replace it with the recombined AttB/AttP site (AttL).

The Ai90D allele is therefore TIGRE-frt3-Ins-TRE-LSL-Chronos/EGFP-WPRE-bGHpA-Ins-AttL.

The resulting Ai90D mice were bred with C57BL/6J wildtype mice for several generations (and the PhiC31 gene was removed). In 2015, black males from generation N6F1 were sent to The Jackson Laboratory Repository. Upon arrival, sperm was cryopreserved. To establish our live colony, frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

The Chronos/EGFP fusion protein, designed by Dr. Ed Boyden (MIT), is composed of a mammalian codon-optimized Stigeoclonium helveticum-derived channelrhodopsin ShChR (Chronos) fused in-frame via a 18 nucleotide linker to the N-terminus of enhanced green fluorescent protein (EGFP). Chronos is a blue-to-green light-driven (~470-530 nm) cation channel that depolarizes the cell and causes action potentials. Chronos has high light sensitivity and faster kinetics than previous channelrhodopsins.

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 TetR or rTetR binding to tetO.

Expression Data

Expressed Gene	GFP, Green Fluorescent Protein,
Expressed Gene	COP4, Channelrhodopsin, Chlamydomonas
Site of Expression	Expression of GFP is under the control of tetO in cells/tissues where promoters driving Cre recombinase are expressed.

Control Suggestions

Approximate Controls

000664 C57BL/6J

Additional Information

Considerations for Choosing Controls

Selected References

Madisen L; Garner AR; Shimaoka D; Chuong AS; Klapoetke NC; Li L; van der Bourg A; Niino Y; Egolf L; Monetti C; Gu H; Mills M; Cheng A; Tasic B; Nguyen TN; Sunkin SM; Benucci A; Nagy A; Miyawaki A; Helmchen F; Empson RM; Knopfel T; Boyden ES; Reid RC; Carandini M; Zeng H. 2015. Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance. Neuron 85(5):942-58PubMed: 25741722 MGI: J:219930

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Genetics

Igs7^{tm90.1(tetO-COP4/EGFP)Hze}*

Allele Symbol: Igs7^{tm90.1(tetO-COP4/EGFP)Hze}*

Allele Name	targeted mutation 90.1, Hongkui Zeng
Allele Type	Targeted (Conditional ready (e.g. floxed), Reporter, Inducible)
Allele Synonym(s)	Ai90(TITL-Chronos)-D; Ai90(TITL-ShChR)-D; Ai90D; Ai90deltaHygro
Gene Symbol and Name	Igs7, intergenic site 7
Gene Synonym(s)
Promoter	tetO, tet operator,
Expressed Gene	GFP, Green Fluorescent Protein,
Expressed Gene	COP4, Channelrhodopsin, Chlamydomonas
Site of Expression	Expression of GFP is under the control of tetO in cells/tissues where promoters driving Cre recombinase are expressed.
Strain of Origin	(129S6/SvEvTac x C57BL/6NCrl)F1
Chromosome	9
Molecular Note	The targeting vector was designed with (from 5' to 3') an FRT3 site, two tandem copies of the chicken beta-globin HS4 insulator element (to reduce reporter gene expression in absence of transactivator protein), a modified Tet response element (TRE or tetO), a loxP-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), the COP4/EGFP coding sequence, a WPRE sequence (to enhance mRNA transcript stability), a bovine growth hormone polyA signal, two tandem copies of the chicken beta-globin HS4 insulator element, an AttB site, a PGK-5' hygro cassette, an RNA splice donor and an FRT5 site.The COP4/EGFP fusion protein is composed of a mammalian codon-optimized Stigeoclonium helveticum-derived channelrhodopsin ShChR (Chronos) fused in-frame via a 18 nucleotide linker to the N-terminus of enhanced green fluorescent protein (EGFP). Chronos is a blue-to-green light-driven (~470-530 nm) cation channel that depolarizes the cell and causes action potentials. Chronos has high light sensitivity and faster kinetics than previous channelrhodopsins. Embryonic stem cells, previously targeted with FRT3-AttB-PGK-neoR-polyA::FRT5::splice acceptor::3' hygro cassette::SV40 polyA: AttP in the same locus, were re-transfected with the targeting vector and Flp recombinase vector. Mutant mice were bred with Gt(ROSA)26Sor^{tm3(phiC31*)Sor} mice to remove to remove the AttB/AttP-flanked PGK-hygro-SV40pA cassette and replace it with the recombined AttB/AttP site (AttL). The Ai90D allele is therefore TIGRE-frt3-Ins-TRE-LSL-Chronos/EGFP-WPRE-bGHpA-Ins-AttL.

Disease/Phenotype

Disease Terms

Research Areas By Phenotype

This mouse can be used to support research in many areas including:

Research Tools
- Diabetes and Obesity Research
  - loxP
- Genetics Research
  - Mutagenesis and Transgenesis
  - Tissue/Cell Markers: cell marker for bone marrow transplantation
  - Tissue/Cell Markers: transplantation marker for embryonic and adult tissue
  - Mutagenesis and Transgenesis: Cre-lox System
  - Tissue/Cell Markers
  - Tissue/Cell Markers: Cre-lox System
  - Mutagenesis and Transgenesis: Tetop Tet System
- Cre-lox System
  - loxP-flanked Sequences
  - loxP-flanked Sequences: Test/Reporter
- Neurobiology Research
  - Tetop Tet System
- Developmental Biology Research
  - transplantation marker for embryonic and adult tissue
  - Cre-lox System
- Reproductive Biology Research
  - transplantation marker for embryonic and adult tissue
  - Cre-lox System
  - Tetop Tet System
- Fluorescent Proteins
- Cancer Research
  - Tetop Tet System
- Cardiovascular Research
  - Tetop Tet System
- Tet Expression Systems
  - tTA/rtTA Responsive Strains
Neurobiology Research
- Cre-lox System
  - loxP-flanked Sequences
  - loxP-flanked Sequences: Test/Reporter
- Optogenetic and Chemogenetic tools
  - Channelrhodopsin expressing strains
  - Cre-dependent optogenetic tools
- Fluorescent protein expression in neural tissue
- Tet Expression System
  - tTA/rtTA Responsive Strains

Mammalian Phenotype Terms by Genotype

The following phenotype relates to a compound genotype created using this strain

Genotype: Igs7^{tm90.1(tetO-COP4/EGFP)Hze}/Igs7⁺
involves: 129S4/SvJaeSor 129S6/SvEvTac * C57BL/6

normal phenotype

no abnormal phenotype detected
- mice are viable and fertile

References

Madisen L; Garner AR; Shimaoka D; Chuong AS; Klapoetke NC; Li L; van der Bourg A; Niino Y; Egolf L; Monetti C; Gu H; Mills M; Cheng A; Tasic B; Nguyen TN; Sunkin SM; Benucci A; Nagy A; Miyawaki A; Helmchen F; Empson RM; Knopfel T; Boyden ES; Reid RC; Carandini M; Zeng H. 2015. Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance. Neuron 85(5):942-58PubMed: 25741722 MGI: J:219930

Additional References

Klapoetke NC; Murata Y; Kim SS; Pulver SR; Birdsey-Benson A; Cho YK; Morimoto TK; Chuong AS; Carpenter EJ; Tian Z; Wang J; Xie Y; Yan Z; Zhang Y; Chow BY; Surek B; Melkonian M; Jayaraman V; Constantine-Paton M; Wong GK; Boyden ES. 2014. Independent optical excitation of distinct neural populations. Nat Methods 11(3):338-46PubMed: 24509633 MGI: J:207450

Additional - Igs7^{tm90.1(tetO-COP4/EGFP)Hze}* related

Technical Support

CONTACT TECHNICAL SUPPORT

Genotyping Protocols
- Standard PCR:Igs7
- Genotyping resources and troubleshooting
Breeding Considerations

When maintaining a live colony, heterozygous mice may be bred to wildtype mice from the colony or to C57BL/6J inbred mice (Stock No. 000664). The donating investigator has not attempted to generate homozygous mice to date (March 2014).
- Additional Breeding and Husbandry Support
Citation
When using the Ai90(TITL-Chronos)-D or Ai90D mouse strain in a publication, please cite the originating article(s) and include JAX stock #024100 in your Materials and Methods section.

Animal Health Reports

Facility Barrier Level Descriptions

Production of mice from cryopreserved embryos or sperm occurs in a maximum barrier room, G200

Pricing & Availability

Cryo Recovery

Domestic
International

Live Mouse

Age

Genotype

Price

weeks

Cryorecovery - Not-For-Profit & Academic Pricing

Service/Product	Description	Price
	Heterozygous or Wildtype forIgs7<tm90.1(tetO-COP4*/EGFP)Hze>

Related Products and Services

Frozen Mouse Embryo	B6.Cg-Igs7<tm90.1(tetO-COP4*/EGFP)Hze>/J	$2595.00
Frozen Mouse Embryo	B6.Cg-Igs7<tm90.1(tetO-COP4*/EGFP)Hze>/J	$2595.00
Frozen Mouse Embryo	B6.Cg-Igs7<tm90.1(tetO-COP4*/EGFP)Hze>/J	$3373.50
Frozen Mouse Embryo	B6.Cg-Igs7<tm90.1(tetO-COP4*/EGFP)Hze>/J	$3373.50

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Also Known As:Ai90(TITL-Chronos)-D or Ai90D

Donating Investigator

Genetic overview

Research Applications

Base Price

Detailed Description

Development

Expression Data

Control Suggestions

Approximate Controls

Additional Information

Selected References

Igs7tm90.1(tetO-COP4*/EGFP)Hze

Allele Symbol: Igs7tm90.1(tetO-COP4*/EGFP)Hze

Disease Terms

Research Areas By Phenotype

This mouse can be used to support research in many areas including:

Mammalian Phenotype Terms by Genotype

Genotype: Igs7tm90.1(tetO-COP4*/EGFP)Hze/Igs7+involves: 129S4/SvJaeSor * 129S6/SvEvTac * C57BL/6

normal phenotype

References

Additional References

Additional - Igs7tm90.1(tetO-COP4*/EGFP)Hze related

Genotyping Protocols

Breeding Considerations

Citation

Animal Health Reports

Production of mice from cryopreserved embryos or sperm occurs in a maximum barrier room, G200

Live Mouse

Cryorecovery - Not-For-Profit & Academic Pricing

Related Products and Services

Payment Terms and Conditions

The Jackson Laboratory's Genotype Promise

Terms Of Use

Additional Use Restrictions Apply

Licensing Information

JAX® Mice, Products & Services Conditions of Use

No Warranty

Credit for PRODUCTS or SERVICES

Animal Care and Use for SERVICES

No Liability

Igs7^{tm90.1(tetO-COP4/EGFP)Hze}*

Allele Symbol: Igs7^{tm90.1(tetO-COP4/EGFP)Hze}*

Genotype: Igs7^{tm90.1(tetO-COP4/EGFP)Hze}/Igs7⁺
involves: 129S4/SvJaeSor 129S6/SvEvTac * C57BL/6

Additional - Igs7^{tm90.1(tetO-COP4/EGFP)Hze}* related