B6;SJL-Tg(Thy1-COP4*H134R/EYFP)20Gfng/J

Stock number: 012350
Product name: Thy1-mhChR2-EYFP Line 20
Research areas: Neurobiology Research, Research Tools, Sensorineural Research

Description

Thy1-mhChR2-YFP transgenic mice express an improved channelrhodopsin-2/EYFP fusion protein (mhChR2::YFP) directed to neural cells by the modified murine Thy1 promoter region. 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. These transgenic mice can be used in optogenetic studies for in vivo control of motor behavior by addition or removal of blue light.

Detailed description

Mice hemizygous for the Thy1-mhChR2-YFP transgene are viable and fertile with expression of the mhChR2::YFP fusion protein directed to neural cells by the modified murine Thy1 promoter region. The mhChR2::YFP fusion protein is composed of a mammalian codon-optimized Chlamydomonas reinhardtii-derived channelrhodopsin-2 (optimized with an N-terminal beta2 nictinic acytlcholine receptor signal peptide and C-terminal ER-export and Golgi-export motifs) that harbors a gain-of-function H134R substitution 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 specifically reports that Thy1-mhChR2-YFP mice derived from founder line 20 exhibit high EGFP expression in cortex, CA1, thalamus, superior colliculus, inferior culliculus, brainstem, amygdala and cerebellum. These Thy1-mhChR2-YFP line 20 transgenic mice may be useful 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, or for fluorescent labeling of neural tissues.

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), 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-mhChR2-YFP transgene was designed in the laboratory of Dr. Guoping Feng (Duke University). A channelrhodopsin-2 cDNA sequence derived from green alga Chlamydomonas reinhardtii was modified for optimal mammalian expression (beta2 nictinic acytlcholine receptor signal peptide (MAGHSNSMALFSFSLLWLCSGVLGTEF) at the N-terminus, and ER-exporting motif (RSRFVKKDGHCNVQFINVGEKGQRYLA) and Golgi-exporting motif (NSFCYENEVALTSK) (both from potassium channel Kir2.1 gene) at the C-terminus) and modified with a gain-of-function H134R substitution (CAC to CGC) designed to cause larger stationary photocurrents. This B2SP-mhChR2-ER-Golgi sequence (also called B2SP-hChR2-H134R-ER export-Golgi export) was fused in-frame to the amino terminus of an enhanced yellow fluorescent protein sequence (EYFP) via a Not1 site with GCGGCCGCC linker sequence. The resulting mhChR2::YFP fusion protein (also called hChR2-H134R-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-B2SP-mhChR2-EYFP-ER-Golgi-BGH polyA (also called Thy1-B2SP-hChR2-H134R-EYFP-ER export-Golgi export-BGH polyA) transgene was injected into B6SJLF1 fertilized oocytes. Transgenic founders were bred with C57BL/6J to generate the Thy1-mhChR2-YFP line 20 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. Upon arrival, mice were bred to C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish the colony.

Allele

Symbol: Tg(Thy1-COP4*H134R/EYFP)20Gfng
Name: transgene insertion 20, Guoping Feng
MGI accession ID: MGI:4454792
Generation method: Transgenic
Attributes: Reporter
Synonyms: Thy1-mhChR2-EYFP Line 20; Thy1-mhChR2-YFP Line 20
Marker symbol: Tg(Thy1-COP4*H134R/EYFP)20Gfng
Marker name: transgene insertion 20, Guoping Feng
Chromosome: UN
Strain of origin: (C57BL/6 x SJL)F1
Expressed genes: YFP,Yellow Fluorescent Protein,jellyfish; COP4,Channelrhodopsin,Chlamydomonas
Site of expression: Transgenic mice express an improved channelrhodopsin-2/EYFP fusion protein (mhChR2::YFP) directed to neural cells.
Molecular note: A channelrhodopsin-2 cDNA sequence derived from green alga Chlamydomonas reinhardtii was modified for optimal mammalian expression (beta2 nictinic acytlcholine receptor signal peptide (MAGHSNSMALFSFSLLWLCSGVLGTEF) at the N-terminus, and ER-exporting motif (RSRFVKKDGHCNVQFINVGEKGQRYLA) and Golgi-exporting motif (NSFCYENEVALTSK) (both from potassium channel Kir2.1 gene) at the C-terminus) and modified with a gain-of-function H134R substitution (CAC to CGC) designed to cause larger stationary photocurrents. This B2SP-mhChR2-ER-Golgi sequence (also called B2SP-hChR2-H134R-ER export-Golgi export) was fused in-frame to the amino terminus of an enhanced yellow fluorescent protein sequence (EYFP) via a Not1 site with GCGGCCGCC linker sequence. The resulting mhChR2::YFP fusion protein (also called hChR2-H134R-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. Mice derived from founder line 20 exhibit high EGFP expression in cortex, CA1, thalamus, superior colliculus, inferior culliculus, brainstem, amygdala and cerebellum.