B6.Cg-Gt(ROSA)26Sor^{tm32(CAG-COP4*H134R/EYFP)Hze}/J

Stock number: 024109
Product name: Ai32 or Ai32(RCL-ChR2(H134R)/EYFP)
Research areas: Neurobiology Research, Research Tools

Description

Ai32 mice express an improved channelrhodopsin-2/EYFP fusion protein following exposure to Cre recombinase. These mice can be used in optogenetic studies for rapid in vivo activation of excitable cells by illumination with blue light (450-490 nm).

Detailed description

Ai32 mice homozygous for the Rosa-CAG-LSL-ChR2(H134R)-EYFP-WPRE conditional allele are viable and fertile. A loxP-flanked STOP cassette prevents transcription of the downstream ChR2(H134R)-EYFP fusion gene. Because this CAG promoter driven reporter construct was targeted for insertion into the Gt(ROSA)26Sor locus, ChR2(H134R)-EYFP expression is determined by which tissue(s) express Cre recombinase.

When bred to mice that express Cre recombinase, the resulting offspring will have the STOP cassette deleted in the cre-expressing tissues; resulting in expression of the ChR2(H134R)-EYFP fusion protein. ChR2(H134R)-EYFP expression following exposure to cre can be detected by EYFP fluorescence (and presumably by mRNA [in situ hybridization] and antibody staining [immunohistochemistry]; although this was not tested by the donating investigator).

The donating investigator reports Ai32 mice have no significant expression of ChR2(H134R)-EYFP prior to introduction of Cre recombinase. Importantly, very low levels of ChR2(H134R)-EYFP expression may be present before Cre recombination - but the ChR2(H134R)-EYFP expression levels after Cre recombination are significantly greater than those baseline levels. As such, it is recommended that researchers include Cre-negative Ai32 controls to establish the baseline ChR2(H134R)-EYFP levels in their experiments.

For characterization information, see images at the Allen Institute for Brain Science website (Ai32 images).

Of note, the FRT sites flanking the mutation allow for additional targeted replacement of the reporter sequences through Flp-mediated recombination if so desired. Similarly, the attB/attP-flanked selection cassette may be removed by introduction of the site-specific bacteriophage PhiC31 integrase if so desired.

The ChR2(H134R)-EYFP fusion protein is composed of a Chlamydomonas reinhardtii-derived channelrhodopsin-2 that harbors a gain-of-function H134R substitution fused in-frame with an enhanced yellow fluorescent protein. The ChR2(H134R) is designed to cause larger stationary photocurrents compared to ChR2.
The bacterial opsins are retinal-binding proteins that combine a light-sensitive domain with an ion channel or pump; providing light-dependent ion transport, membrane potential alteration, and sensory functions to bacteria. This ChR2(H134R) functions as a blue light-driven cation channel that depolarizes the cell and causes action potentials. As such, illuminating ChR2(H134R)-expressing cells with blue light (450-490 nm) leads to rapid and reversible photostimulation of action potential firing activity in these cells.

Development

The Rosa-CAG-LSL-ChR2(H134R)-EYFP-WPRE targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), an FRT site, a loxP-flanked STOP cassette (with stop codons in all 3 reading frames and a triple polyA signal), a ChR2(H134R)-EYFP fusion gene, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a BGH polyA signal, and an attB/attP-flanked PGK-FRT-Neo-polyA cassette. To create the ChR2(H134R)-EYFP fusion gene, a cDNA sequence encoding the first 315 amino acids of channelrhodopsin-2 (derived from the green alga Chlamydomonas reinhardtii) was modified with a gain-of-function H134R substitution (CAC to CGC) designed to cause larger stationary photocurrents. This ChR2(H134R) sequence was fused in-frame to the amino terminus of an enhanced yellow fluorescent protein sequence, resulting in the final ChR2(H134R)-EYFP fusion protein sequence. This entire construct was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus via electroporation of (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells. Correctly targeted ES cells (clone Ai32) were selected. Chimeric mice were bred with C57BL/6 wildtype mice and/or PhiC31-expressing mice (C57BL/6J congenic background; see Stock No. 007743) - with some resulting offspring retaining the attB/attP-flanked cassette (more detail below). Mice with the Ai32 allele (Rosa-CAG-LSL-ChR2(H134R)-EYFP-WPRE-BGHpA-attB-PGK-FRT-Neo-pA-attP) were then bred with C57BL/6J wildtype mice for at least one additional generation prior to sending to The Jackson Laboratory Repository in 2010 as Stock No. 012569. Upon arrival, some Ai32 mice were backcrossed with C57BL/6J inbred mice (Stock No. 000664) for five additional generations to establish this C57BL/6-congenic strain as Stock No. 024109. This colony shows 99.5%-100% of 183 SNP markers throughout the genome are C57BL/6 allele-type.

All of the mice originally sent to The Jackson Laboratory Repository, as well as several generations of breeding Ai32 mutant mice with C57BL/6J inbred mice, have resulted in mice that genotype as completely co-segregating for the neo cassette and YFP-WPRE portion of the Ai32 construct. As such, these mice harbor the Ai32 mutation (retaining the attB/attP-flanked PGK-FRT-Neo-polyA cassette) rather than the Ai32Δneo mutation.

Allele

Symbol: Gt(ROSA)26Sor^{tm32(CAG-COP4*H134R/EYFP)Hze}
Name: targeted mutation 32, Hongkui Zeng
MGI accession ID: MGI:5013789
Generation method: Targeted
Attributes: Reporter
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Chromosome: 6
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: COP4,Channelrhodopsin,Chlamydomonas
Site of expression: Cre excision of the stop signal results in expression of an improved channelrhodopsin-2/ EYFP fusion protein (hChR2(H134R)- EYFP) in cre-expressing tissues.
Molecular note: The Rosa-CAG-LSL-ChR2(H134R)-EYFP-WPRE targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), an FRT site, a loxP-flanked STOP cassette (with stop codons in all 3 reading frames and a triple polyA signal), a ChR2(H134R)-EYFP fusion gene, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a BGH polyA signal, and an attB/attP-flanked PGK-FRT-Neo-polyA cassette. To create the ChR2(H134R)-EYFP fusion gene, a cDNA sequence encoding the first 315 amino acids of channelrhodopsin-2 (derived from the green alga Chlamydomonas reinhardtii) was modified with a gain-of-function H134R substitution (CAC to CGC) designed to cause larger stationary photocurrents. This ChR2(H134R) sequence was fused in-frame to the amino terminus of an enhanced yellow fluorescent protein sequence, resulting in the final ChR2(H134R)-EYFP fusion protein sequence. This entire construct was inserted between exons 1 and 2 of the locus.