B6;129S-Gt(ROSA)26Sor^tm2.1Ksvo/J

Stock number: 024846
Product name: R26 LSL FSF ReaChR-mCitrine
Research areas: Neurobiology Research, Research Tools

Description

ReaChR protein (a red-shifted channelrhodopsin) was tagged with mCitrine fluorescent protein and placed under the conditional control of the Gt(ROSA)26Sor promoter. Excision of two stop cassettes (one flanked by loxP sites, another flanked by FRT sites) enables fluorescent expression. As an optogenetic tool, this strain is useful for the selective activation and mapping of neuron populations.

Anecdotal reports suggest that the FRT-flanked Stop cassette may* be showing FRT-site read through in this line, effectively making this a cre-dependent strain only.

Detailed description

Optogenetics enables the controlled excitation of defined neuron populations in animals that have been genetically sensitized to light. The ReaChR channelrhodopsin variant used in such studies incorporates portions of VChR1 and VChR2 (channelrhodopsins from Volvox carteri), ChIEF (the transmembrane domains of Chlamydomonas reinhardtii channelrhodopsins 1 and 2 modified with an Ile to Val mutation), and a point mutation (L171I) to enhance activation by red light (~590 nm). Axons expressing the protein become selectively inactivated during continuous excitation.

Although ReaChR viral vector models for neurological circuit mapping exist, they demonstrate some variability in expression around the injection site. To achieve more uniform expression, ReaChR tagged with fluorescent protein mCitrine was integrated into the mouse genome. Conditional expression from the Gt(ROSA)26Sor promoter is enabled upon excision of both loxP- and Frt-flanked stop cassettes. If bred to ROSA26-Cre (e.g. Stock# ) or ROSA26-Flp mice (e.g. Stock No. 003946) to ubiquitously remove one of the two conditional stop cassettes, expression of ReaChR in offspring can be made dependent on the introduction of a single recombinase directed at the remaining stop cassette.

Progeny of animals crossed with a ROSA26-Flp strain that are injected with AAV-Syn-iCre (viral vector Cre) in the primary somatosensory cortex (S1) demonstrate fluorescence at the injection site as well as in S1-originating exons extending to the primary motor cortex (M1).

In vitro recording of coronal sections from Flp-excised animals infected with AAV-Syn-iCre (virus expressing iCre from a synapsin promoter) reveal uniform current responses from L2/3 pyramidal neurons stimulated with 590 nm light.

Applications for cortical circuit mapping with dual photostimulation (2CRACM) have been demonstrated.

Anecdotal reports suggest that the FRT-flanked Stop cassette may* be showing FRT-site read through in this line, effectively making this a cre-dependent strain only.

Development

A CAG promoter (cytomegalovirus (CMV) enhancer fused to the chicken beta-actin promoter), FRT-flanked 3PA STOP cassette, loxP-flanked 3PA STOP cassette, ReaChR red-shifted channelrhodopsin, fluorescent reporter mCitrine, woodchuck hepatitis post-transcriptional regulatory element (WPRE), polyA (pA) tail, and AttB/AttP-flanked neomycin resistance cassette were introduced to intron 1 of the Gt(ROSA)26Sor gene via targeted recombination in C57BL/6J x 129S6 F1-derived embryonic stem (ES) cells. Resultant animals were bred to ROSA26-PhiC31 mice (backcrossed N10 onto C57BL/6J; see Stock No. 007743), to remove the AttB/AttP-flanked neomycin cassette and replace it with the AttB/AttP recombined site (AttL). The resulting "Rosa26 Cag fsf lsl ReaChR-citrine" mice were backcrossed to C57BL/6J for two generations by the donating laboratory prior to sending to The Jackson Laboratory Repository in 2014 as Stock No. 024846.

Anecdotal reports suggest that the FRT-flanked Stop cassette may* be showing FRT-site read through in this line, effectively making this a cre-dependent strain only.

Allele

Symbol: Gt(ROSA)26Sor^tm2.1Ksvo
Name: targeted mutation 2.1, Keral Svoboda
MGI accession ID: MGI:5605717
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Chromosome: 6
Strain of origin: (C57BL/6J x 129S6/SvEvTac)F1
Expressed genes: YFP,Yellow Fluorescent Protein,jellyfish
Site of expression: The fluorescent protein mCitrine will be expressed in cells/tissues where promoters driving Cre recombinase and Flp recombinase are expressed.
Molecular note: A CAG promoter (cytomegalovirus (CMV) enhancer fused to the chicken beta-actin promoter), FRT-flanked stop cassette, loxP-flanked stop cassette, ReaChR red-shifted channelrhodopsin, fluorescent reporter mCitrine, woodchuck hepatitis post-transcriptional regulatory element (WPRE), poly A (pA) tail, and AttB/AttP-flanked neomycin resistance cassette were introduced to intron 1 of the Gt(ROSA)26Sor gene. PhiC31o-mediated recombination removed the neo cassette.