B6;SJL-Tg(Thy1-COP3/EYFP)1Gfng/J

Stock number: 012341
Research areas: Neurobiology Research, Research Tools, Sensorineural Research

Description

Thy1-vChR1-EYFP transgenic mice express an improved channelrhodopsin-1/EYFP fusion protein (VChR1-EYFP) directed to neural cells by the modified murine Thy1 promoter region. Illuminating VChR1-expressing neurons with green light (~535 nm) leads to rapid and reversible photostimulation of action potential firing/neural activity in these cells. These transgenic mice can be used for in vivo optogenetic studies to map neural circuit connectivity and spatial distribution of neurons by addition or removal of light.

Detailed description

Mice hemizygous for the Thy1-VChR1-EYFP transgene are viable and fertile with expression of the VChR1-EYFP fusion protein directed to neural cells by the modified murine Thy1 promoter region.

The donating investigator specifically reports that Thy1-VChR1-EYFP mice derived from founder line 1 (Thy1-VChR1-EYFP line 1) exhibit exhibit medium and sparse labeling of EGFP at cortex, hippocampus (CA1, CA3 and dentate gyrus), thalamus, superior colliculus, inferior culliculus, brainstem and amydagala. These Thy1-VChR1-EYFP line 1 transgenic mice may be useful for rapid control of motor behavior by addition or removal of light, for ex vivo and in vivo mapping of neural circuit connectivity and spatial distribution of neurons following illumination, or for fluorescent labeling of neural tissues.

For transgenic mice from different founder lines (lines 4 and 8 [Stock Nos. 012344 and 012348, respectively]), the VChR1-EYFP fusion protein is efficiently targeted to the plasma membrane and is largely free of unwanted aggregation/retention within the endoplasmic reticulum (some intracellular VChR1 aggregation is reported within the lateroposterior thalamic nuclei). Also for lines 4 and 8, photoactivation is more effective with age at least out to 23 days of age: this age-dependent increase in the degree of photoactivation presumably results from higher levels of VChR1 expression during this time.

The VChR1-EYFP fusion protein is composed of a synthetic, mammalian codon-optimized, red-shifted channelrhodopsin-1 derived from Volvox carteri (VChR1) fused in-frame with an enhanced yellow fluorescent protein (EYFP). Compared with ChR2, VChR1 is more light-sensitive and has a markedly (~70 nm) red-shifted action spectrum with a maximum at ~535 nm (green light). While VChR1 is quite effective in depolarizing and firing pyramidal neurons, its slow deactivation kinetics limit the ability to precisely control the timing of action potential firing. To accommodate the slower deactivation kinetics of VChR1, the time interval between photostimuli should be set to allow time for responses to fully recover between stimuli.

The bacterial opsins are retinal-binding proteins that provide light-dependent ion transport and sensory functions to a family of halophilic bacteria; and this VChR1 functions as a green light-driven cation channel that depolarizes the cell and causes action potentials. As such, illumination of VChR1-expressing neurons leads to rapid and reversible photostimulation of action potential firing/neural activity in these cells.

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 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 5 (Stock No. 014545), 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 Thy1-VChR1-YFP (Thy1-VChR1-EYFP) transgene was designed in the laboratory of Dr. Guoping Feng (Duke University). A fusion protein was created with a synthetic, mammalian codon-optimized, red-shifted channelrhodopsin-1 cDNA sequence derived from Volvox carteri (VChR1; GenBank accession number EU622855 [residues 1-300]) fused in-frame to the amino terminus of an enhanced yellow fluorescent protein sequence (EYFP). This enhanced fusion protein (VChR1-EYFP) was placed downstream of the modified regulatory region of the "murine thy1.2 gene" (extending from the promoter to the intron following exon 4, excluding exon 3 and its flanking introns), and followed by a bovine growth hormone polyA signal. The resulting Thy1-VChR1-EYFP transgene was injected into B6SJLF1 fertilized oocytes. Transgenic founders were bred with C57BL/6J to generate the Thy1-VChR1-EYFP line 1 colony. The colony was backcrossed to C57BL6/J mice for a total of at least two generations prior to arrival at The Jackson Laboratory Repository in 2010. 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).

Allele

Symbol: Tg(Thy1-COP3/EYFP)1Gfng
Name: transgene insertion 1, Guoping Feng
MGI accession ID: MGI:4454790
Generation method: Transgenic
Attributes: Reporter
Synonyms: Thy1-vChR1-EYFP Line 1; Thy1-vChR1-YFP line 1
Marker symbol: Tg(Thy1-COP3/EYFP)1Gfng
Marker name: transgene insertion 1, Guoping Feng
Chromosome: UN
Strain of origin: (C57BL/6 x SJL)F1
Expressed genes: YFP,Yellow Fluorescent Protein,jellyfish
Site of expression: Transgenic mice express an improved channelrhodopsin-1/EYFP fusion protein (VChR1::YFP) directed to neural cells.
Molecular note: A fusion protein was created with a synthetic, mammalian codon-optimized, red-shifted channelrhodopsin-1 cDNA sequence derived from Volvox carteri (VChR1; GenBank accession number EU622855 [residues 1-300]) fused in-frame to the amino terminus of an enhanced yellow fluorescent protein sequence (EYFP). This enhanced fusion protein (VChR1::YFP) was placed downstream of the modified regulatory region of the "murine thy1.2 gene" (extending from the promoter to the intron following exon 4, excluding exon 3 and its flanking introns), and followed by a bovine growth hormone polyA signal. Mice derived from founder line 1 exhibit medium and sparse labeling of EGFP at cortex, CA1, CA3 and dentate gyrus of hippocampus, thalamus, superior colliculus, inferior culliculus, brainstem, amydagala.