B6;129-Gt(ROSA)26Sor^{tm1(CAG-COP4*E123T*H134R,-tdTomato)Gfng}/J

Stock number: 017455
Product name: R26-CAG-LSL-2XChETA-tdTomato
Research areas: Neurobiology Research, Research Tools

Description

The R26-CAG-LSL-2XChETA-tdTomato reporter allele is designed to express two individual proteins following exposure to Cre recombinase; the ChR2-E123T accelerated (ChETA)/H134R variant of channelrhodopsin-2 and tdTomato fluorescent protein gene. When used with neuron-specific Cre recombinase expressing strains, the double mutant mice can be used in optogenetic studies and visualization of whole neurons.

Detailed description

The R26-CAG-LSL-2XChETA-tdTomato reporter allele has two copies of the ChR2-E123T accelerated (ChETA)/H134R variant of channelrhodopsin-2, a P2A oligopeptide sequence (to mediate ribosomal skipping), and the tdTomato fluorescent protein gene, all inserted into the Gt(ROSA)26Sor locus. Expression of ChETA/H134R and tdTomato is blocked by a loxP-flanked STOP fragment placed between the ChETA/H134R sequence and tdTomato sequence and the Gt(ROSA)26Sor promoter. This strain serves as a reporter strain, with successful Cre recombinase excision being indicated by ChETA/H134R and tdTomato expression in cre-expressing tissues. When crossed with a neuron-specific Cre recombinase expressing strain, functional ChETA/H134R and tdTomato expression is observed in all brain regions tested thus far. ChETA/H134R and tdTomato are expressed separately and not as fusion proteins. The Donating Investigator reports that mice that are homozygous for the targeted mutation are viable, fertile, normal in size and do not display any gross physical or behavioral abnormalities.

Development

The R26-CAG-LSL-2XChETA-tdTomato targeting vector was designed with (from 5' to 3') the CAG promoter, a loxP site, a PGK-NEO-pA cassette (STOP), a second loxP site, two copies of a channelrhodopsin-2 (COP4) variant harboring the E123T and H134R mutations (ChR2-E123T accelerated (ChETA)/H134R or ChETA/H134R), a P2A oligopeptide sequence (to mediate ribosomal skipping), the tdTomato variant of enhanced red fluorescent protein, and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability). The construct was inserted into the Gt(ROSA)26Sor locus via electroporation into (129X1/SvJ x 129S1/Sv)F1-Kitl⁺-derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into receipient blastocysts. The resulting chimeric animals were crossed to C57BL/6J mice. Upon arrival at The Jackson Laboratory, the mice were crossed to C57BL/6J (Stock No. 000664) at least once to establish the colony.

Allele

Symbol: Gt(ROSA)26Sor^{tm1(CAG-COP4*E123T*H134R,-tdTomato)Gfng}
Name: targeted mutation 1, Guoping Feng
MGI accession ID: MGI:5301821
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: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Expressed genes: COP4,Channelrhodopsin,Chlamydomonas
Site of expression: Cre excision of the stop signal results in expression of a channelrhodopsin-2 (COP4) and tdTomato in cre-expressing tissues.
Molecular note: A targeting vector, which contained the CAG promoter, a loxP site flanked STOP fragment and pGK-NEO-pA cassette, 2 copies of channelrhodopsin-2 with E123T mutation (ChETA variant) and the H134R mutation, P2A-tdTomato variant of Enhanced Red Fluorescent Protein, and WPRE (Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element) sequence to enhance expression level, was inserted into the locus.