B6.Cg-Tg(Chat-COP4*H134R/EYFP,Slc18a3)5Gfng/J

Stock number: 014545
Research areas: Neurobiology Research, Research Tools, Sensorineural Research

Description

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.

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.

Allele

Symbol: Tg(Chat-COP4*H134R/EYFP,Slc18a3)5Gfng
Name: transgene insertion 5, Guoping Feng
MGI accession ID: MGI:4950474
Generation method: Transgenic
Attributes: Reporter
Marker symbol: Tg(Chat-COP4*H134R/EYFP,Slc18a3)5Gfng
Marker name: transgene insertion 5, Guoping Feng
Chromosome: UN
Strain of origin: (C57BL/6 x CBA)F1
Expressed genes: COP4,Channelrhodopsin,Chlamydomonas; YFP,Yellow Fluorescent Protein,jellyfish
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.
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).