Overview

Also Known As: Ai32 or Ai32(RCL-ChR2(H134R)/EYFP)

Ai32 mice express an improved channelrhodopsin-2/EYFP fusion protein following exposure to Cre recombinase. These mice can be used in optogenetic studies for rapid in vivo activation of excitable cells by illumination with blue light (450-490 nm).

Donating Investigator

Hongkui Zeng, Allen Institute for Brain Science

Genetic overview

Genetic Background	Generation
	?+N9F12 (2019-05-06 00:00:00)

Gt(ROSA)26Sor^{tm32(CAG-COP4H134R/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

Neurobiology Research
Research Tools

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Base Price

Starting at:

$255.00 Domestic price for female 4-week

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Details

Detailed Description

Ai32 mice homozygous for the Rosa-CAG-LSL-ChR2(H134R)-EYFP-WPRE conditional allele are viable and fertile. A loxP-flanked STOP cassette prevents transcription of the downstream ChR2(H134R)-EYFP fusion gene. Because this CAG promoter driven reporter construct was targeted for insertion into the Gt(ROSA)26Sor locus, ChR2(H134R)-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 ChR2(H134R)-EYFP fusion protein. ChR2(H134R)-EYFP expression following exposure to cre can be detected by EYFP fluorescence (and presumably by mRNA [in situ hybridization] and antibody staining [immunohistochemistry]; although this was not tested by the donating investigator).

The donating investigator reports Ai32 mice have no significant expression of ChR2(H134R)-EYFP prior to introduction of Cre recombinase. Importantly, very low levels of ChR2(H134R)-EYFP expression may be present before Cre recombination - but the ChR2(H134R)-EYFP expression levels after Cre recombination are significantly greater than those baseline levels. As such, it is recommended that researchers include Cre-negative Ai32 controls to establish the baseline ChR2(H134R)-EYFP levels in their experiments.

For characterization information, see images at the Allen Institute for Brain Science website (Ai32 images).

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.

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.
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. 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 Rosa-CAG-LSL-ChR2(H134R)-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), a ChR2(H134R)-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. 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. This entire construct 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 Ai32) were selected. Chimeric mice were bred with C57BL/6 wildtype mice and/or PhiC31-expressing mice (C57BL/6J congenic background; see Stock No. 007743) - with some resulting offspring retaining the attB/attP-flanked cassette (more detail below). Mice with the Ai32 allele (Rosa-CAG-LSL-ChR2(H134R)-EYFP-WPRE-BGHpA-attB-PGK-FRT-Neo-pA-attP) were then bred with C57BL/6J wildtype mice for at least one additional generation prior to sending to The Jackson Laboratory Repository in 2010 as Stock No. 012569. Upon arrival, some Ai32 mice were backcrossed with C57BL/6J inbred mice (Stock No. 000664) for five additional generations to establish this C57BL/6-congenic strain as Stock No. 024109. This colony shows 99.5%-100% of 183 SNP markers throughout the genome are C57BL/6 allele-type.

All of the mice originally sent to The Jackson Laboratory Repository, as well as several generations of breeding Ai32 mutant mice with C57BL/6J inbred mice, have resulted in mice that genotype as completely co-segregating for the neo cassette and YFP-WPRE portion of the Ai32 construct. As such, these mice harbor the Ai32 mutation (retaining the attB/attP-flanked PGK-FRT-Neo-polyA cassette) rather than the Ai32Δneo mutation.

Expression Data

Expressed Gene	COP4, Channelrhodopsin, Chlamydomonas
Site of Expression	Cre excision of the stop signal results in expression of an improved channelrhodopsin-2/ EYFP fusion protein (hChR2(H134R)- EYFP) in cre-expressing tissues.

Control Suggestions

Approximate Controls

000664 C57BL/6J

Additional Information

Considerations for Choosing Controls

Selected References

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

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Genetics

Gt(ROSA)26Sor^{tm32(CAG-COP4H134R/EYFP)Hze}*

Allele Symbol: Gt(ROSA)26Sor^{tm32(CAG-COP4H134R/EYFP)Hze}*

Allele Name	targeted mutation 32, Hongkui Zeng
Allele Type	Targeted (Reporter)
Allele Synonym(s)	targeted mutation 32, Hongkui Zeng; Gt(ROSA)26Sor^{tm32(CAG-COP4*H134R/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	COP4, Channelrhodopsin, Chlamydomonas
Site of Expression	Cre excision of the stop signal results in expression of an improved channelrhodopsin-2/ EYFP fusion protein (hChR2(H134R)- EYFP) in cre-expressing tissues.
Strain of Origin	(129S6/SvEvTac x C57BL/6NCrl)F1
Chromosome	6
Molecular Note	The Rosa-CAG-LSL-ChR2(H134R)-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), a ChR2(H134R)-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. 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. This entire construct was inserted between exons 1 and 2 of the 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:

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

Mammalian Phenotype Terms by Genotype

The following phenotype information is associated with a similar, but not exact match to this JAX^® Mice strain

Genotype: Gt(ROSA)26Sor^{tm32(CAG-COP4*H134R/EYFP)Hze}/Gt(ROSA)26Sor⁺
involves: 129S6/SvEvTac

mammalian phenotype

no phenotypic analysis

References

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 References

Schulz JM; Knoflach F; Hernandez MC; Bischofberger J. 2018. Dendrite-targeting interneurons control synaptic NMDA-receptor activation via nonlinear alpha5-GABAA receptors. Nat Commun 9(1):3576PubMed: 30177704 MGI: J:267792
Antoine MW; Langberg T; Schnepel P; Feldman DE. 2019. Increased Excitation-Inhibition Ratio Stabilizes Synapse and Circuit Excitability in Four Autism Mouse Models. Neuron 101(4):648-661.e4PubMed: 30679017 MGI: J:271893

Additional - Gt(ROSA)26Sor^{tm32(CAG-COP4H134R/EYFP)Hze}* related

Technical Support

Contact Technical Support

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

When maintaining a live colony, homozygous mice may be bred together.
- Additional Breeding and Husbandry Support
Mating System
- Homozygote x Homozygote
Citation
When using the Ai32 or Ai32(RCL-ChR2(H134R)/EYFP) mouse strain in a publication, please cite the originating article(s) and include JAX stock #024109 in your Materials and Methods section.

Animal Health Reports

Facility Barrier Level Descriptions

AX10 (Standard)

Pricing & Availability

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Age

Genotype

Price

weeks

Cryorecovery - Not-For-Profit & Academic Pricing

Service	Genotype	Price
	Heterozygous for Gt(ROSA)26Sor<tm32(CAG-COP4*H134R/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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The Jackson Laboratory has rigorous genetic quality control and mutant gene genotyping programs to ensure the genetic background of JAX® Mice strains as well as the genotypes of strains with identified molecular mutations. JAX® Mice strains are only made available to researchers after meeting our standards. However, the phenotype of each strain may not be fully characterized and/or captured in the strain data sheets. Therefore, we cannot guarantee a strain's phenotype will meet all expectations. To ensure that JAX® Mice will meet the needs of individual research projects or when requesting a strain that is new to your research, we suggest ordering and performing tests on a small number of mice to determine suitability for your particular project.

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Also Known As: Ai32 or Ai32(RCL-ChR2(H134R)/EYFP)

Donating Investigator

Genetic overview

Research Applications

Base Price

Detailed Description

Development

Expression Data

Control Suggestions

Approximate Controls

Additional Information

Selected References

Gt(ROSA)26Sortm32(CAG-COP4*H134R/EYFP)Hze

Allele Symbol: Gt(ROSA)26Sortm32(CAG-COP4*H134R/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)26Sortm32(CAG-COP4*H134R/EYFP)Hze/Gt(ROSA)26Sor+involves: 129S6/SvEvTac

mammalian phenotype

References

Additional References

Additional - Gt(ROSA)26Sortm32(CAG-COP4*H134R/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^{tm32(CAG-COP4H134R/EYFP)Hze}*

Allele Symbol: Gt(ROSA)26Sor^{tm32(CAG-COP4H134R/EYFP)Hze}*

Genotype: Gt(ROSA)26Sor^{tm32(CAG-COP4*H134R/EYFP)Hze}/Gt(ROSA)26Sor⁺
involves: 129S6/SvEvTac

Additional - Gt(ROSA)26Sor^{tm32(CAG-COP4H134R/EYFP)Hze}* related