Optogenetics enables the selective activation of genetically-targeted neuronal populations using light-sensitive ion channels. Genetic strategies using Cre-dependent mouse strains, especially the Ai32 line expressing Channelrhodopsin (ChR2)-EYFP fusion protein, have been a popular means to drive opsin expression in a cell-type specific manner. Here we report a low level of leaky 'off-target' (Cre-independent) ChR2-EYFP expression in Ai32/Ai32 homozygous mice throughout the nervous system. This leaky off-target expression was characterized in multiple prevalent nervous system regions using anti-EYFP immunostaining. Expression of full-length ChR2-EYFP protein was confirmed using immunoprecipitation followed by Western blotting. Notably, light stimulation of these ChR2-EYFP expressing neurons in the spinal cord dorsal horn did not induce detectable photocurrents in juvenile 4-week old mice. Given the wide use of the Ai32 line by many labs, our results suggest researchers should be vigilant of possible off-target ChR2-EYFP expression in their region of interest, especially when generating Ai32/Ai32 homozygotes to drive high levels of ChR2-EYFP expression in adult mice.
Pubmed ID: 30913225 RIS Download
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These Brainbow 1.0 (founder line L) mice allow labeling of individual neuronal types (specifically hippocampal neuron cell bodies, and including motor neurons, dentate gyrus granule cells, pyramidal neurons of the cortex and CA1 area) with approximately 166 distinguishable color variations in cre recombined cells, and may also be useful in conjunction with other Brainbow strains (Stock No. 007901, Stock No. 007911, Stock No. 007921) for neurobiological studies. These Thy1-Brainbow 1.0 (line L) transgenic mice are viable and fertile. The mice possess multiple fluorescent protein sequences uniquely flanked with pairs of incompatible Lox sites alternated to create mutually exclusive recombination events; allowing stochastic expression of multiple fluorescent proteins from a single transgene. Prior to Cre-mediated recombination, the fluorescent protein immediately adjacent to the promoter, dTomato (RFP), is expressed in peripheral and central neurons. When bred to Cre recombinase expressing mice, the resulting offspring can have one of three expression outcomes for each transgene in each cell of the cre expressing tissue(s): dTomato (RFP) (no recombination), mCerulean (CFP), or mYFP. Integration of tandem transgene copies yields combinatorial fluorescent protein expression in each cell, and thus many possible cell colors, providing a way to distinguish adjacent neurons and visualize other cellular interactions. Of note, the single FRT site inserted in the transgene allows tandem transgene copy number reduction through Flp-mediated recombination if desired. These Brainbow 1.0 (founder line L) mice were found to have multiple transgene copies that allow labeling of individual neuronal types (specifically hippocampal neuron cell bodies, and including motor neurons, dentate gyrus granule cells, pyramidal neurons of the cortex and CA1 area) with approximately 166 distinguishable color variations in cre recombined cells, and may also be useful in conjunction with other Brainbow strains (Stock No. 007901, Stock No. 007911, Stock No. 007921) for neurobiological studies. This mouse can be used to support research in many areas including:
Neurobiology Research
* Cre-lox System (loxP-flanked Sequences)
* Fluorescent protein expression in neural tissue
Research Tools
* Cre-lox-System (loxP-flanked Sequences: Test/Reporter)
* Developmental Biology Research (Cre-lox system)
* Developmental Biology Research (transplantation marker for embryonic and adult tissue)
* FLP-FRT System (FRT-flanked Sequences)
* Fluorescent Proteins * Genetics Research (Mutagenesis and Transgenesis: Cre-lox system) * Genetics Research (Tissue/Cell Markers: Cre-lox system) * Genetics Research (Tissue/Cell Markers: astrocyte-specific marker) * Genetics Research (Tissue/Cell Markers: astrocytes) * Genetics Research (Tissue/Cell Markers: astrocytes, neurons) * Genetics Research (Tissue/Cell Markers: glial cells) * Genetics Research (Tissue/Cell Markers: multiple) * Genetics Research (Tissue/Cell Markers: neurons) * Genetics Research (Tissue/Cell Markers: transplantation marker for embryonic and adult tissue) * Neurobiology Research (astrocyte-specific marker) * Neurobiology Research (cell marker) * YFP related Research Tools * Fluorescent Proteins Control: 000664 C57BL/6J (approximate)
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View all literature mentionsThis polyclonal targets Goat IgG
View all literature mentionsThis polyclonal targets GFP
View all literature mentionsThis polyclonal targets Green Fluorescent Protein (GFP)
View all literature mentionsThis polyclonal targets Glial Fibrillary Acidic Protein (GFAP)
View all literature mentionsMus musculus with name B6;129P2-Omptm4(cre)Mom/MomJ from IMSR.
View all literature mentionsMus musculus with name B6.Cg-Gt(ROSA)26Sortm32(CAG-COP4*H134R/EYFP)Hze/J from IMSR.
View all literature mentionsThis polyclonal targets GFP
View all literature mentionsThis polyclonal targets Green Fluorescent Protein (GFP)
View all literature mentionsMus musculus with name B6.Cg-Gt(ROSA)26Sortm32(CAG-COP4*H134R/EYFP)Hze/J from IMSR.
View all literature mentionsMus musculus with name B6;129P2-Omptm4(cre)Mom/MomJ from IMSR.
View all literature mentionsThis polyclonal targets Goat IgG
View all literature mentionsThis polyclonal targets GFP
View all literature mentionsThis polyclonal targets Green Fluorescent Protein (GFP)
View all literature mentionsThis polyclonal targets Glial Fibrillary Acidic Protein (GFAP)
View all literature mentionsMus musculus with name B6;129P2-Omptm4(cre)Mom/MomJ from IMSR.
View all literature mentionsMus musculus with name B6.Cg-Gt(ROSA)26Sortm32(CAG-COP4*H134R/EYFP)Hze/J from IMSR.
View all literature mentions