B6.Cg-Gt(ROSA)26Sor^{tm80.1(CAG-COP4*L132C/EYFP)Hze}/J

Stock number: 025109
Product name: Ai80(RCFL-CatCh)-D or Ai80D
Research areas: Neurobiology Research, Research Tools

Description

The Ai80(RCFL-CatCh)-D (or Ai80D) conditional allele has both an frt-flanked STOP cassette and a loxP-flanked STOP cassette preventing transcription of the calcium translocating channelrhodopsin (CatCh; an L132C mutant channelrhodopsin with enhanced Ca2+ permeability, accelerated response kinetics and ~70-fold greater light sensitivity) and EYFP. After removal of the flanked STOP cassettes by FLP- and Cre-recombination, CatCh/EYFP expression is expected in cells/tissues expressing both FLP recombinase and Cre recombinase. Subsequent illumination of CatCh-expressing cells with blue-to-green light leads to reversible photostimulation of action potential firing/neural activity in these cells.

Detailed description

Ai80(RCFL-CatCh)-D mice (also Ai80D) harbor the Rosa-CAG-FrtSTOPFrt-LoxSTOPLox-CatCh/EYFP conditional allele designed with both an frt-flanked STOP cassette and a loxP-flanked STOP cassette preventing transcription of the CatCh/EYFP fusion gene (ChR2(L132C)/EYFP; the calcium translocating, L132C mutant channelrhodopsin variant (CatCh) fused in-frame with an enhanced yellow fluorescent protein). Although under control of the endogenous Gt(ROSA)26Sor promoter/enhancer regions and the CAG hybrid promoter, widespread expression of CatCh/EYFP is prevented by both STOP cassettes. After removal of both flanked STOP cassettes via Flp- and cre-mediated recombination, expression of CatCh/EYFP is expected in cells/tissues where the expression patterns of the individual promoters driving FLP recombinase and Cre recombinase overlap.

Specifically, the donating investigator reports that Ai80D mice have no CatCh/EYFP expression (EGFP fluorescence) prior to introduction of both FLP and Cre recombinase. Weak, membrane-localized EYFP fluorescence is observed after deletion of both STOP cassettes. The donating investigator also reports that order in which the flanked STOP cassettes are removal is arbitrary. Heterozygous mice are viable and fertile with no reported gross physical or behavioral abnormalities.

These Ai80D mice have not been fully characterized to date (September 2014). Whether CatCh/EYFP expression levels are measurable by mRNA (in situ hybridization) or antibody staining (immunohistochemistry) is not yet known. Functional testing has not yet been performed to test if light-induced opsin activation occurs at levels sufficient to effectively depolarize/activate cortical neurons. The donating investigator has not attempted to generate homozygous mice.

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. Chlamydomonas reinhardtii-derived channelrhodopsin-2 (ChR2 or cop4) is a blue light-driven cation channel that depolarizes the cell and causes action potentials. The calcium translocating channelrhodopsin (CatCh) is a ChR2 variant modified to have an L132C amino acid substitution for enhanced Ca2+ permeability; resulting in accelerated response time and a voltage response that is ~70-fold more light sensitive than wildtype ChR2. As such, illuminating CatCh-expressing cells with blue-to-green light (~450-530 nm; max 474 nm) leads to rapid and reversible photostimulation of robust action potential firing activity in these cells. Because CatCh activation is possible at naturally occurring light intensities while maintaining high temporal precision, it may also have applications in gene-therapeutic visual restoration efforts.

Development

These Ai80(RCFL-CatCh)-D mice (also called Ai80D) have both an frt-flanked STOP cassette and a loxP-flanked STOP cassette upstream of the CatCh/EYFP fluorescent protein, all inserted into the Gt(ROSA)26Sor locus. The specific details are below.
The Rosa-pCAG-FSF-LSL-CatCh/EYFP-WPRE-bGHpA-AttB-PGK-Neo-polyA-AttP targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), an frt-flanked STOP cassette (with a short open reading frame, followed by translational stops in all three reading frames, a synthetic polyA sequence, a polyA sequence from the human growth hormone gene, and a polyA sequence from the Herpes Simplex Virus TK gene), a loxP-flanked STOP cassette (with stop codons in all three reading frames linked to three repeats of the SV40 polyA signal), a sequence encoding the CatCh/EYFP fluorescent protein (described below), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a bovine growth hormone polyA signal, an AttB site, a PGK promoter-Neomycin resistance gene-PGK polyA cassette, and an AttP site. This entire targeting vector 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 were selected. Chimeric female was bred to PhiC31-expressing male (C57BL/6J congenic background; see Stock No. 007743) to remove the AttB/AttP-flanked PGK-Neo-polyA cassette and replace it with the recombined AttB/AttP site (AttL). The resulting Ai80D mice were bred with C57BL/6J wildtype mice for several generations (and the PhiC31 gene was removed) prior to sending generation N7F1 males to The Jackson Laboratory Repository in 2014. Upon arrival, males were used to cryopreserve sperm. To establish the living Ai80D mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).

To create the CatCh/EYFP (ChR2(L132C)/EYFP) fusion protein, a cDNA sequence encoding the first 309 amino acids of green alga Chlamydomonas reinhardtii channelrhodopsin-2 (ChR2 or cop4) was modified with an L132C amino acid substitution (CTC-to-TGC) designed to cause enhanced Ca2+ permeability. This calcium translocating channelrhodopsin (ChR2(L132C) or CatCh) sequence was followed by a proline residue and 12 nucleotide linker, and then fused in-frame to the amino terminus of an enhanced yellow fluorescent protein (EYFP) sequence, resulting in the final CatCh/EYFP fusion protein sequence.

Allele

Symbol: Gt(ROSA)26Sor^{tm80.1(CAG-COP4*L132C/EYFP)Hze}
Name: targeted mutation 80.1, Hongkui Zeng
MGI accession ID: MGI:5607578
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: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: COP4/EYFP,Channelrhodopsin-2 (Chlamydomonas reinhardtii) and Enhanced Yellow Fluorescent Protein fusion gene,
Site of expression: After removal of both flanked STOP cassettes via Flp- and cre-mediated recombination, expression of CatCh/EYFP is expected in cells/tissues where the expression patterns of the individual promoters driving FLP recombinase and Cre recombinase overlap.
Molecular note: The targeting vector was designed with (from 5' to 3')a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), an frt-flanked STOP cassette (with a short open reading frame, followed by translational stops in all three reading frames, a synthetic polyA sequence, a polyA sequence from the human growth hormone gene, and a polyA sequence from the Herpes Simplex Virus TK gene), a loxP-flanked STOP cassette (with stop codons in all three reading frames linked to three repeats of the SV40 polyA signal), a sequence encoding the CatCh/EYFP fluorescent protein, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a bovine growth hormone polyA signal, an AttB site, a PGK promoter-Neomycin resistance gene-PGK polyA cassette, and an AttP site. This entire targeting vector was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus. Chimeric female mice was bred to mice expressing Gt(ROSA)26Sor^{tm3(phiC31*)Sor} to remove the AttB/AttP-flanked PGK-Neo-polyA cassette and replace it with the recombined AttB/AttP site (AttL).