Overview

ChAT-ChR2-YFP BAC transgenic mice may be useful in optogenetic studies for rapid control of motor behavior by addition or removal of light, for ex vivo and in vivo studies of neural circuitry/connectivity following illumination, for fluorescent labeling of cholinergic neuronal populations, or for studying the consequences of overactive cholinergic signaling in information processing, memory, behavior and physical fitness.

Donating Investigator

Guoping Feng, Massachusetts Institute of Technology

Genetic overview

Genetic Background	Generation

Tg(Chat-COP4*H134R/EYFP,Slc18a3)5Gfng

Allele Type
Transgenic (Reporter)

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Research Applications

Research Tools
Neurobiology Research
Sensorineural Research

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Base Price

Starting at:

$2,854.50 Domestic price Cryo Recovery

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Details

Important Note

The vesicular acetylcholine transporter gene (Slc18a3 or VAChT) within the Chat locus on the BAC transgene was not disrupted. The donating investigator reports that these ChAT-mhChR2-YFP line 5 mice are expected to have a similar VAChT overexpression phenotype as reported for ChAT-mhChR2-YFP line 6 (Stock No. 014546). That is, VAChT overexpression in hippocampus and brainstem results in increased cholinergic tone; the functional consequence of which is increased physical endurance but severe cognitive deficits in attention, working memory and spatial memory.

Detailed Description

Mice hemizygous for the ChAT-ChR2-YFP BAC transgene are viable and fertile (see note below), with expression of the mhChR2::YFP fusion protein directed to cholinergic neuronal populations by the mouse choline acetyltransferase (Chat or ChAT) promoter/enhancer regions on the BAC transgene. The mhChR2::YFP fusion protein is composed of a mammalian codon-optimized Chlamydomonas reinhardtii-derived channelrhodopsin-2 that was modified to harbor a gain-of-function H134R substitution (mhChR2; also called hChR2-H134R) fused in-frame with an enhanced yellow fluorescent protein (EYFP). The mhChR2 is designed to cause larger stationary photocurrents compared to ChR2. The bacterial opsins are retinal-binding proteins that provide light-dependent ion transport and sensory functions to a family of halophilic bacteria; and this mhChR2 functions as a blue light-driven cation channel that depolarizes the cell and causes action potentials. As such, illuminating mhChR2-expressing neurons with blue light (450-490 nm) leads to rapid and reversible photostimulation of action potential firing/neural activity in these cells.

The donating investigator reports that EYFP expression is only weakly visible by direct fluorescence (epifluorescence microscope), but can be amplified by antibody enhancement in fixed brain sections. ChAT-mhChR2-YFP mice derived from founder line 5 (ChAT-ChR2-YFP line 5) exhibit moderate EYFP expression in striatum, trochlear nucleus, medial habenula, interpeduncular nucleus and various brainstem motor neuron nuclei. Lower EYFP expression is found in cortex, hippocampus, and other brain regions. Functional activation of neurons with blue laser light has only been confirmed in striatal cholinergic neurons in this founder line. ChAT co-staining shows precise co-localization with mhChR2-EYFP expression neurons; suggesting mhChR2-EYFP labeled neurons in this line are cholinergic neurons. Moderate EYFP fluorescence is also observed in the ventral gray horn in transverse section of the spinal cord. The donating investigator also reports that these ChAT-mhChR2-YFP line 5 mice exhibit a similar, but less bright expression pattern as ChAT-mhChR2-YFP line 6 (Stock No. 014546).

Because the vesicular acetylcholine transporter gene (Slc18a3 or VAChT) within the Chat locus on the BAC transgene was not disrupted, VAChT protein is overespressed. The VAChT overespression phenotype is reported for ChAT-mhChR2-YFP line 6 (Stock No. 014546), and the donating investigator expects a similar phenotype for ChAT-mhChR2-YFP line 5 mice. That is, VAChT overexpression in hippocampus and brainstem results in increased cholinergic tone; the functional consequence of which is increased physical endurance but severe cognitive deficits in attention, working memory and spatial memory. ChAT-ChR2-EYFP line 6 mice also consume more food and water during the dark cycle compared to control mice. Compared to wildtype animals, ChAT-ChR2-EYFP line 6 mice show no changes in metabolic profile, locomotor activity in a novel environment, anxiety-like behavior, depression-like behavior, gross sensorimotor function, and ability to use cues to learn a task.

This optogenetic strain is one of many from the same transgene creator/donating investigator with light-inducible neurobiology applications; including
Thy1-ChR2-YFP line 18 (Stock No. 007612),
Thy1-ChR2-YFP line 9 (Stock No. 007615),
Thy1-eNpHR-YFP line 2 (Stock No. 012332),
Thy1-eNpHR-YFP line 4 (Stock No. 012334),
Thy1-vChR1-YFP line 1 (Stock No. 012341),
Thy1-vChR1-YFP line 4 (Stock No. 012344),
Thy1-vChR1-YFP line 8 (Stock No. 012348),
Thy1-mhChR2-YFP Line 20 (Stock No. 012350),
Prv-mhChR2-YFP Line 15 (Stock No. 012355),
ChAT-ChR2-YFP line 6 (Stock No. 014546),
VGAT-ChR2-YFP line 8 (Stock No. 014548),
and TpH2-ChR2-YFP line 5 (Stock No. 014555).

Development

The ChAT-mhChR2-YFP transgene (or ChAT-ChR2-YFP BAC transgene) was designed in the laboratory of Dr. Guoping Feng (Massachusetts Institute of Technology). First, a channelrhodopsin-2 cDNA sequence derived from green alga Chlamydomonas reinhardtii was modified to harbor codons optimized for mammalian expression and a gain-of-function H134R substitution (CAC to CGC) designed to cause larger stationary photocurrents. This mhChR2 sequence (also called hChR2-H134R) was fused in-frame to the amino terminus of an enhanced yellow fluorescent protein sequence (EYFP) via a Not1 site with GCGGCCGCC linker sequence. This mhChR2::YFP fusion protein (also called hChR2-H134R-EYFP) sequence was inserted into the coding region of the mouse choline acetyltransferase (Chat or ChAT) locus on the RP23-246B12 bacterial artificial chromosome (BAC) via homologous recombination. This was designed to disable transcription from the Chat locus, and none of the other loci on the BAC were altered. The BAC also includes the vesicular acetylcholine transporter gene (Slc18a3 or VAChT) that, because it is located in the intron between the first and second exons of the Chat gene, is under the control of the Chat promoter. The resulting ~220 kb ChAT-mhChR2-YFP BAC transgene was injected into B6SJLF1 fertilized oocytes. Transgenic founders were bred with C57BL/6J to generate the ChAT-mhChR2-YFP line 5 colony (also called ChAT-ChR2-YFP line 5). The colony was backcrossed to C57BL/6J mice for a total of at least four generations prior to sending to The Jackson Laboratory Repository in 2011. Upon arrival, sperm was cryopreserved. To generate the living colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J female mice (Stock No. 000664). To maintain our live ChAT-ChR2-YFP line 5 colony, hemizygotes are bred with wildtype (noncarrier) mice from the colony or with C57BL/6J inbred mice. Of note, the donating investigator may not have fixed the Y chromosome to the C57BL/6J genetic background during backcrossing.

Expression Data

Expressed Gene	YFP, Yellow Fluorescent Protein, jellyfish
Expressed Gene	COP4, Channelrhodopsin, Chlamydomonas
Site of Expression	EYFP expression in cholinergic neurons of the striatum, trochlear nucleus, medial habenula, interpeduncular nucleus and various brainstem motor neuron nuclei. Lower EYFP expression is found in cortex, hippocampus, and other brain regions.

Control Suggestions

Noncarrier

Approximate Controls

Additional Information

Considerations for Choosing Controls

Selected References

Zhao S; Ting JT; Atallah HE; Qiu L; Tan J; Gloss B; Augustine GJ; Deisseroth K; Luo M; Graybiel AM; Feng G. 2011. Cell type-specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function. Nat Methods 8(9):745-52PubMed: 21985008 MGI: J:175525

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Genetics

Tg(Chat-COP4*H134R/EYFP,Slc18a3)5Gfng

Allele Symbol: Tg(Chat-COP4*H134R/EYFP,Slc18a3)5Gfng

Allele Name	transgene insertion 5, Guoping Feng
Allele Type	Transgenic (Reporter)
Allele Synonym(s)	transgene insertion 5, Guoping Feng; Tg(Chat-COP4*H134R/EYFP,Slc18a3)5Gfng
Gene Symbol and Name	Tg(Chat-COP4*H134R/EYFP,Slc18a3)5Gfng, transgene insertion 5, Guoping Feng
Gene Synonym(s)
Promoter	Chat, choline acetyltransferase, mouse, laboratory
Expressed Gene	YFP, Yellow Fluorescent Protein, jellyfish
Expressed Gene	COP4, Channelrhodopsin, Chlamydomonas
Site of Expression	EYFP expression in cholinergic neurons of the striatum, trochlear nucleus, medial habenula, interpeduncular nucleus and various brainstem motor neuron nuclei. Lower EYFP expression is found in cortex, hippocampus, and other brain regions.
Strain of Origin	(C57BL/6 x CBA)F1
Chromosome	UN
Molecular Note	Transgene expression of the mhChR2::YFP fusion protein is directed to cholinergic neuronal populations by the mouse choline acetyltransferase (Chat or ChAT) promoter/enhancer regions on the BAC transgene. The mhChR2::YFP fusion protein is composed of a mammalian codon-optimized Chlamydomonas reinhardtii-derived channel rhodopsin-2 that was modified to harbor a gain-of-function H134R substitution (mhChR2; also called hChR2-H134R) fused in-frame with an enhanced yellow fluorescent protein (EYFP). The BAC used to generate the construct includes the vesicular acetylcholine transporter gene (Slc18a3 or VAChT). Slc18a3 is located in the intron between the first and second exons of the Chat gene and is under the control of the Chat promoter. The mhChR2 is designed to cause larger stationary photocurrents compared to ChR2. The bacterial opsins are retinal-binding proteins that provide light-dependent ion transport and sensory functions to a family of halophilic bacteria; and this mhChR2 functions as a blue light-driven cation channel that depolarizes the cell and causes action potentials. As such, illuminating mhChR2-expressing neurons with blue light (450-490 nm) leads to rapid and reversible photostimulation of action potential firing/neural activity in these cells. EYFP expression is visible by direct fluorescence (epifluorescence microscope). ChAT-mhChR2-YFP mice derived from founder line 5 exhibit moderate EYFP expression in striatum, trochlear nucleus, medial habenula, interpeduncular nucleus and various brainstem motor neuron nuclei. Lower EYFP expression is found in cortex, hippocampus, and other brain regions. ChAT co-staining shows precise co-localization with mhChR2-EYFP expression neurons; suggesting mhChR2-EYFP labeled neurons in this line are cholinergic neurons. Moderate EYFP fluorescence is also observed in the ventral gray horn in transverse section of the spinal cord. The donating investigator also reports that these ChAT-mhChR2-YFP line 5 mice exhibit a similar, but less bright expression pattern as ChAT-mhChR2-YFP line 6 (JAX Stock No. 014546).
Mutations Made By	Guoping Feng, Massachusetts Institute of Technology

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: neurons
  - Tissue/Cell Markers
- Neurobiology Research
  - cell marker
- Sensorineural Research
- Fluorescent Proteins
Neurobiology Research
- Optogenetic and Chemogenetic tools
  - Channelrhodopsin expressing strains
- Fluorescent protein expression in neural tissue
Sensorineural Research

Mammalian Phenotype Terms by Genotype

References

Zhao S; Ting JT; Atallah HE; Qiu L; Tan J; Gloss B; Augustine GJ; Deisseroth K; Luo M; Graybiel AM; Feng G. 2011. Cell type-specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function. Nat Methods 8(9):745-52PubMed: 21985008 MGI: J:175525

Additional - Tg(Chat-COP4*H134R/EYFP,Slc18a3)5Gfng related

Technical Support

Contact Technical Support

Genotyping Protocols
- Standard PCR:Tg(Chat-COP4*H134R/EYFP)
- Standard PCR:Tg(Chat-COP4*H134R/EYFP)
- Genotyping resources and troubleshooting
Breeding Considerations

When maintaining a live colony, hemizygous mice may be bred with wildtype (noncarrier) mice from the colony or with C57BL/6J inbred mice (Stock No. 000664).
- Additional Breeding and Husbandry Support
Citation
When using the B6.Cg-Tg(Chat-COP4*H134R/EYFP,Slc18a3)5Gfng/J mouse strain in a publication, please cite the originating article(s) and include JAX stock #014545 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 - Pricing

Service	Genotype	Price
	Hemizygous or Non carrier for Tg(Chat-COP4*H134R/EYFP)5Gfng

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.

Related Products and Services

Frozen Mouse Embryo	B6.Cg-Tg(Chat-COP4*H134R/EYFP Slc18a3)5Gfng/J Frozen Embryo	$2595.00
Frozen Mouse Embryo	B6.Cg-Tg(Chat-COP4*H134R/EYFP Slc18a3)5Gfng/J Frozen Embryo	$2595.00
Frozen Mouse Embryo	B6.Cg-Tg(Chat-COP4*H134R/EYFP Slc18a3)5Gfng/J Frozen Embryo	$3373.50
Frozen Mouse Embryo	B6.Cg-Tg(Chat-COP4*H134R/EYFP Slc18a3)5Gfng/J Frozen Embryo	$3373.50

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Donating Investigator

Genetic overview

Research Applications

Base Price

Important Note

Detailed Description

Development

Expression Data

Control Suggestions

Approximate Controls

Additional Information

Selected References

Tg(Chat-COP4*H134R/EYFP,Slc18a3)5Gfng

Allele Symbol: Tg(Chat-COP4*H134R/EYFP,Slc18a3)5Gfng

Disease Terms

Research Areas By Genotype

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

Mammalian Phenotype Terms by Genotype

References

Additional - Tg(Chat-COP4*H134R/EYFP,Slc18a3)5Gfng 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 - Pricing

Related Products and Services

Payment Terms and Conditions

The Jackson Laboratory's Genotype Promise

Terms of Use

Licensing Information

JAX® Mice, Products & Services Conditions of Use

No Warranty

Credit for PRODUCTS or SERVICES

Animal Care and Use for SERVICES

No Liability