B6.Cg-Tg(Slc32a1-COP4*H134R/EYFP)8Gfng/J

Stock number: 014548
Product name: VGAT-ChR2-EYFP line 8
Research areas: Neurobiology Research, Research Tools, Sensorineural Research

Description

Mice carrying this transgene (also known as VGAT-ChR2-YFP BAC) 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, and for fluorescent labeling of GABAergic interneuronal populations.

Detailed description

Mice hemizygous for this transgene (also known as VGAT-ChR2-YFP BAC transgene) are viable and fertile, with expression of the mhChR2::YFP fusion protein directed to GABAergic interneuronal populations by the mouse vesicular GABA transporter (VGAT or Slc32a1) 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 visible by direct fluorescence (epifluorescence microscope). VGAT-ChR2-YFP mice derived from founder line 8 exhibit strong EYFP expression in olfactory bulb (glomerular and mitral cell layers), thalamic reticular nucleus, superior colliculus, inferior colliculus, brainstem, and molecular layer of cerebellum. Moderate EYFP expression is found in cortex, hippocampus, thalamus, and granule cell layer of olfactory bulb. GAD67 co-staining shows the mhChR2-EYFP expressing neurons are GABAergic interneurons. High EYFP fluorescence is also observed for gray matter in transverse section of the spinal cord.

These VGAT-ChR2-YFP BAC 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 GABAergic interneuronal populations.

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 5 (Stock No. 014545), ChAT-ChR2-YFP line 6 (Stock No. 014546), and TpH2-ChR2-YFP line 5 (Stock No. 014555).

Development

The VGAT-mhChR2-YFP BAC transgene (or VGAT-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 solute carrier family 32 [GABA vesicular transporter] member 1 (Slc32a1 or VGAT) locus on the RP23-392P11 bacterial artificial chromosome (BAC) via homologous recombination. This was designed to disable transcription from the Slc32a1 locus, and none of the other loci on the BAC were altered. The resulting ~196 kbp VGAT-mhChR2-YFP BAC transgene was injected into B6SJLF1 fertilized oocytes. Transgenic founders were bred with C57BL/6J to generate the VGAT-mhChR2-YFP line 8 colony (also called VGAT-ChR2-YFP line 8). The colony was backcrossed to C57BL/6J mice for a total of at least five 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 VGAT-ChR2-YFP line 8 colony, hemizygotes are bred with wildtype (noncarrier) mice from the colony or with C57BL/6J inbred mice. There are approximately 20 copies of the transgene present. Of note, the donating investigator may not have fixed the Y chromosome to the C57BL/6J genetic background during backcrossing.

Allele

Symbol: Tg(Slc32a1-COP4*H134R/EYFP)8Gfng
Name: transgene insertion 8, Guoping Feng
MGI accession ID: MGI:4950481
Generation method: Transgenic
Attributes: Reporter
Marker symbol: Tg(Slc32a1-COP4*H134R/EYFP)8Gfng
Marker name: transgene insertion 8, Guoping Feng
Chromosome: UN
Strain of origin: (C57BL/6 x SJL)F1
Expressed genes: COP4,Channelrhodopsin,Chlamydomonas; YFP,Yellow Fluorescent Protein,jellyfish
Site of expression: Transgenic mice express an improved channelrhodopsin-2/EYFP fusion protein (mhChR2::YFP) directed to GABAergic interneuronal populations.
Molecular note: Transgene expression of the mhChR2::YFP fusion protein is directed to GABAergic interneuronal populations by the mouse vesicular GABA transporter (VGAT or Slc32a1) 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. EYFP expression is visible by direct fluorescence (epifluorescence microscope). VGAT-mhChR2-YFP mice derived from founder line 8 exhibit strong EYFP expression in olfactory bulb (glomerular and mitral cell layers), thalamic reticular nucleus, superior colliculus, inferior colliculus, brainstem, and molecular layer of cerebellum. Moderate EYFP expression is found in cortex, hippocampus, thalamus, and granule cell layer of olfactory bulb. GAD67 co-staining shows the mhChR2-EYFP expressing neurons are GABAergic interneurons. High EYFP fluorescence is also observed for gray matter in transverse section of the spinal cord.