Overview

Also Known As: Ai39 or Ai39(RCL-eNpHR3.0/EYFP)

These Ai39 mice conditionally express an improved Halorhodopsin/EYFP fusion protein from the endogenous Gt(ROSA)26Sor locus. Following Cre-mediated removal of the STOP cassette, EYFP expression is observed in the cre-expressing cells. Subsequent illumination of these cells with yellow-to-red light leads to reversible photoinhibition of action potential firing/neural activity in these cells. These Ai39 mice are useful for optogenetic studies to express an inhibitory opsin that effectively silences the activity of cortical neurons.

Donating Investigator

Hongkui Zeng, Allen Institute for Brain Science

Genetic overview

Genetic Background	Generation
	F?N3pN1+F19 (2018-12-14 00:00:00)

Gt(ROSA)26Sor^{tm39(CAG-hop/EYFP)Hze}

Allele Type	Gene Symbol	Gene Name
Targeted (Reporter)	Gt(ROSA)26Sor	gene trap ROSA 26, Philippe Soriano

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Research Applications

Research Tools
Neurobiology Research

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Base Price

Starting at:

$255.00 Domestic price for female

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Details

Detailed Description

Ai39 mice heterozygous for the Rosa-CAG-LSL-eNpHR3.0-EYFP-WPRE conditional allele are viable and fertile. A loxP-flanked STOP cassette prevents transcription of the downstream eNpHR3.0-EYFP fusion gene (see below for detailed description of eNpHR3.0-EYFP). Because the CAG promoter driven reporter construct was targeted for insertion into the Gt(ROSA)26Sor locus, eNpHR3.0-EYFP expression is determined by which tissue(s) express Cre recombinase. When bred to mice that express Cre recombinase, the resulting offspring will have the STOP cassette deleted in the cre-expressing tissues; resulting in expression of the eNpHR3.0-EYFP fusion protein. The donating investigator reports that Ai39 mice do not express eNpHR3.0-EYFP prior to introduction of Cre recombinase.

Fusion protein expression following exposure to cre can be detected by EYFP fluorescence and mRNA (in situ hybridization) [and presumably by antibody staining (immunohistochemistry); although this was not tested by the donating investigator]. Following exposure to Cre recombinase, illuminating eNpHR3.0-expressing neurons with yellow-to-red light leads to reversible photoinhibition of action potential firing/neural activity in these cells. The donating investigator specifically reports that expression of the inhibitory opsin occurs at levels sufficient to effectively silence the activity of cortical neurons. Fusion protein expression in tissues other than brain has not yet been evaluated by the donating investigator (April 2011). The phenotype of homozygous mice has not been characterized by the donating investigator. Homozygous mice are viable and fertile.

Of note, the FRT sites flanking the mutation allow for additional targeted replacement of the reporter sequences through Flp-mediated recombination if so desired. Similarly, the attB/attP-flanked selection cassette may be removed by introduction of the site-specific bacteriophage PhiC31 integrase if so desired.

For characterization information, see images at the Allen Institute for Brain Science website (Ai39 images).

The eNpHR3.0-EYFP fusion protein is composed of the Natronomonas pharaonis-derived halorhodopsin (NpHR) fused in-frame with an enhanced yellow fluorescent protein (EYFP). The eNpHR3.0-EYFP fusion protein has been modified to harbor both the membrane trafficking signal and the endoplasmic reticulum (ER) exporting sequence from the potassium channel Kir2.1 gene. These improvements result in optimized expression in mammalian cells by preventing ER aggregation/enhancing membrane translocation, reducing bleb formation, and enhancing inhibitory capacity. The eNpHR3.0-EYFP fusion protein allows robust photocurrents/action potential inhibition when exposed to light ranging from yellow (~589 nm) to deep red (~660 nm) and at the red/infrared border (~680 nm).

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 third-generation halorhodopsin eNpHR3.0 is an expression-optimized, yellow-to-red light-driven (~580-680 nm), inward chloride ion pump that causes hyperpolarization and prevents action potentials. For example, illumination of NpHR-expressing neurons leads to reversible photoinhibition of action potential firing/neural activity in these cells.

Development

The Rosa-CAG-LSL-eNpHR3.0-EYFP-WPRE targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), an FRT site, a loxP-flanked STOP cassette (with stop codons in all 3 reading frames and a triple polyA signal), an eNpHR3.0-EYFP fusion gene, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a BGH polyA signal, and an attB/attP-flanked PGK-FRT-Neo-polyA cassette.

The NpHR-EYFP fusion protein was first designed with the halorhodopsin from the halophilic bacterium Natronomonas pharaonis (NpHR) fused in-frame with an enhanced yellow fluorescent protein (EYFP). To create the eNpHR3.0-EYFP fusion protein, the NpHR-EYFP fusion protein was modified via addition of a membrane trafficking signal from the potassium channel Kir2.1 gene (amino acids KSRITSEGEYIPLDQIDINV) between NpHR and EYFP, as well as addition of an endoplasmic reticulum (ER) exporting sequence from the potassium channel Kir2.1 gene (amino acids FCYENEV) at the C-terminal of the EYFP. These modifications result in optimized expression in mammalian systems by preventing ER aggregation/enhancing membrane translocation, reducing bleb formation, and enhancing inhibitory capacity. The entire Rosa-CAG-LSL-eNpHR3.0-EYFP-WPRE targeting vector was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus via electroporation of (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells. Correctly targeted ES cells (clone Ai39) were selected.

Chimeric mice were bred with C57BL/6 wildtype mice and/or PhiC31-expressing mice (C57BL/6J congenic background; see Stock No. 007743).

Mutant mice were bred with C57BL/6J wildtype mice for approximately three more generations prior to sending to The Jackson Laboratory Repository. Upon arrival, sperm from Ai39 males was used to fertilize oocytes from C57BL/6J inbred mice (Stock No. 000664) in order to rederive the living colony at The Jackson Laboratory Repository.

All of the mice originally sent to The Jackson Laboratory Repository, as well as all the first generation of rederived offspring, genotype as completely co-segregating for the neo cassette and eNpHR portion of the Ai39 construct. As such, these mice harbor the Ai39 mutation (retaining the attB/attP-flanked PGK-FRT-Neo-polyA cassette) rather than the Ai39Δneo mutation.

As of August 2017, the live colony is ~87-89% C57BL/6.

Expression Data

Expressed Gene	YFP, Yellow Fluorescent Protein, jellyfish
Expressed Gene	HOP, Halorhodopsin, Natronomonas
Site of Expression	Cre excision of the stop signal results in expression of a halorhodopsin/EYFP (eNpHR3.0-EYFP) fusion protein in cre-expressing tissues.

Control Suggestions

Approximate Controls

000664 C57BL/6J

Additional Information

Considerations for Choosing Controls

Selected References

Zeng H. 2011. Direct Database Submission 2011/06/21_Dl1gAf MGI Direct Data SubmissionMGI: J:172634
Madisen L; Mao T; Koch H; Zhuo JM; Berenyi A; Fujisawa S; Hsu YW; Garcia AJ 3rd; Gu X; Zanella S; Kidney J; Gu H; Mao Y; Hooks BM; Boyden ES; Buzsaki G; Ramirez JM; Jones AR; Svoboda K; Han X; Turner EE; Zeng H. 2012. A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing. Nat Neurosci 15(5):793-802PubMed: 22446880 MGI: J:182204

View All References

Genetics

Gt(ROSA)26Sor^{tm39(CAG-hop/EYFP)Hze}

Allele Symbol: Gt(ROSA)26Sor^{tm39(CAG-hop/EYFP)Hze}

Allele Name	targeted mutation 39, Hongkui Zeng
Allele Type	Targeted (Reporter)
Allele Synonym(s)	targeted mutation 39, Hongkui Zeng; Gt(ROSA)26Sor^{tm39(CAG-hop/EYFP)Hze}
Gene Symbol and Name	Gt(ROSA)26Sor, gene trap ROSA 26, Philippe Soriano
Gene Synonym(s)	beta geo; ROSA26; R26; Gtrgeo26; Gtrosa26; Gtrgeo26; Gtrosa26; AV258896; expressed sequence AV258896; gene trap ROSA 26; gene trap ROSA b-geo 26; SETD5-AS1; Thumpd3as1
Expressed Gene	YFP, Yellow Fluorescent Protein, jellyfish
Expressed Gene	HOP, Halorhodopsin, Natronomonas
Site of Expression	Cre excision of the stop signal results in expression of a halorhodopsin/EYFP (eNpHR3.0-EYFP) fusion protein in cre-expressing tissues.
Strain of Origin	(129S6/SvEvTac x C57BL/6NCrl)F1
Chromosome	6
Molecular Note	The Rosa-CAG-LSL-eNpHR3.0-EYFP-WPRE targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), an FRT site, a loxP-flanked STOP cassette (with stop codons in all 3 reading frames and a triple polyA signal), an eNpHR3.0-EYFP fusion gene, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a BGH polyA signal, and an attB/attP-flanked PGK-FRT-Neo-polyA cassette. The NpHR-EYFP fusion protein was first designed with the halorhodopsin from the halophilic bacterium Natronomas pharaonis (NpHR) fused in-frame with an enhanced yellow fluorescent protein (EYFP). To create the eNpHR3.0-EYFP fusion protein, the NpHR-EYFP fusion protein was modified via addition of a membrane trafficking signal from the potassium channel Kir2.1 gene (amino acids KSRITSEGEYIPLDQIDINV) between NpHR and EYFP, as well as addition of an endoplasmic reticulum (ER) exporting sequence from the potassium channel Kir2.1 gene (amino acids FCYENEV) at the C-terminal of the EYFP. These modifications result in optimized expression in mammalian systems by preventing ER aggregation/enhancing membrane translocation, reducing bleb formation, and enhancing inhibitory capacity. The entire Rosa-CAG-LSL-eNpHR3.0-EYFP-WPRE targeting vector was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus.
Mutations Made By	Hongkui Zeng, Allen Institute for Brain Science

Disease/Phenotype

Disease Terms

Research Areas By Genotype

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

Research Tools
- Genetics Research
  - Tissue/Cell Markers
  - Mutagenesis and Transgenesis
  - Tissue/Cell Markers: transplantation marker for embryonic and adult tissue
  - Tissue/Cell Markers: Cre-lox System
  - Mutagenesis and Transgenesis: Cre-lox System
  - Tissue/Cell Markers: cell marker for bone marrow transplantation
- Cre-lox System
  - loxP-flanked Sequences
  - loxP-flanked Sequences: Test/Reporter
- Developmental Biology Research
  - transplantation marker for embryonic and adult tissue
  - Cre-lox System
- Fluorescent Proteins
- Diabetes and Obesity Research
  - loxP
- Reproductive Biology Research
  - transplantation marker for embryonic and adult tissue
  - Cre-lox System
- PhiC31 System
  - attB/attP-flanked Sequences
- FLP-FRT System
  - FRT-flanked Sequences
  - FRT-flanked Sequences: Test/Reporter
Neurobiology Research
- Cre-lox System
  - loxP-flanked Sequences
  - loxP-flanked Sequences: Test/Reporter
- Optogenetic and Chemogenetic tools
  - Cre-dependent optogenetic tools
  - Halorhodopsin expressing strains
- Fluorescent protein expression in neural tissue

Mammalian Phenotype Terms by Genotype

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

Genotype: Gt(ROSA)26Sor^{tm39(CAG-hop/EYFP)Hze}/Gt(ROSA)26Sor⁺
involves: 129S6/SvEvTac * C57BL/6NCr

mammalian phenotype

no phenotypic analysis

References

Zeng H. 2011. Direct Database Submission 2011/06/21_Dl1gAf MGI Direct Data SubmissionMGI: J:172634
Madisen L; Mao T; Koch H; Zhuo JM; Berenyi A; Fujisawa S; Hsu YW; Garcia AJ 3rd; Gu X; Zanella S; Kidney J; Gu H; Mao Y; Hooks BM; Boyden ES; Buzsaki G; Ramirez JM; Jones AR; Svoboda K; Han X; Turner EE; Zeng H. 2012. A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing. Nat Neurosci 15(5):793-802PubMed: 22446880 MGI: J:182204

Additional - Gt(ROSA)26Sor^{tm39(CAG-hop/EYFP)Hze} related

Technical Support

Contact Technical Support

Genotyping Protocols
- Separated PCR: Gt(ROSA)26Sor(eNpHR)
- Genotyping resources and troubleshooting
Dietary Information
- LabDiet® 5K52 formulation (6% fat)
Breeding Considerations

Heterozygous and homozygous mice are viable and fertile. When maintaining a live colony, heterozygous mice may be bred together, to wildtype mice from the colony or to C57BL/6J inbred mice (Stock No. 000664). Alternatively, homozygous mice may be bred together.
- Additional Breeding and Husbandry Support
Mating System
- Homozygote x Homozygote
Citation
When using the Ai39 or Ai39(RCL-eNpHR3.0/EYFP) mouse strain in a publication, please cite the originating article(s) and include JAX stock #014539 in your Materials and Methods section.

Animal Health Reports

Facility Barrier Level Descriptions

FGB29 (Standard)

Pricing & Availability

Available

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Live Mouse

Age

Genotype

Price

weeks

Cryorecovery - Not-For-Profit & Academic Pricing

Service	Genotype	Price
	Heterozygous or Wild-type for Gt(ROSA)26Sor<tm39.1(CAG-HOP/EYFP)Hze>

We will fulfill your order by providing at least two carriers for each strain ordered. The total number, sex, and genotypes provided will vary, although typically 8 or more animals are provided. Please check genotypes which will be recovered. While the genotypes of all animals produced will be communicated to you prior to scheduling shipment, the genotypes of animals provided may not reflect the mating scheme and genotypes described in the strain description. Animals are typically ready to ship in 11-14 weeks. If a second recovery is required to produce the minimum number of animals, then delivery time would increase to approximately 25 weeks. If we fail to produce animals of the correct genotype, you will not be charged. We cannot guarantee the reproductive success of mice shipped to your facility. If the mice are lost after the first three days (post-arrival) or do not produce progeny at your facility, a new order and fee will be necessary.

Cryorecovery to establish a Dedicated Supply for greater quantities of mice. Mice recovered can be used to establish a dedicated colony to contractually supply you mice according to your requirements. Price by quotation.

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Also Known As: Ai39 or Ai39(RCL-eNpHR3.0/EYFP)

Donating Investigator

Genetic overview

Research Applications

Base Price

Detailed Description

Development

Expression Data

Control Suggestions

Approximate Controls

Additional Information

Selected References

Gt(ROSA)26Sortm39(CAG-hop/EYFP)Hze

Allele Symbol: Gt(ROSA)26Sortm39(CAG-hop/EYFP)Hze

Disease Terms

Research Areas By Genotype

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

Mammalian Phenotype Terms by Genotype

Genotype: Gt(ROSA)26Sortm39(CAG-hop/EYFP)Hze/Gt(ROSA)26Sor+involves: 129S6/SvEvTac * C57BL/6NCr

mammalian phenotype

References

Additional - Gt(ROSA)26Sortm39(CAG-hop/EYFP)Hze related

Genotyping Protocols

Dietary Information

Breeding Considerations

Mating System

Citation

Animal Health Reports

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

Gt(ROSA)26Sor^{tm39(CAG-hop/EYFP)Hze}

Allele Symbol: Gt(ROSA)26Sor^{tm39(CAG-hop/EYFP)Hze}

Genotype: Gt(ROSA)26Sor^{tm39(CAG-hop/EYFP)Hze}/Gt(ROSA)26Sor⁺
involves: 129S6/SvEvTac * C57BL/6NCr

Additional - Gt(ROSA)26Sor^{tm39(CAG-hop/EYFP)Hze} related