These Ai39 mice conditionally express an improved Halorhodopsin/EYFP fusion protein from the endogenous Gt(ROSA)26Sor locus. Following Cre-mediated removal of the STOP cassette, EYFP expression is observed in the cre-expressing cells. Subsequent illumination of these cells with yellow-to-red light leads to reversible photoinhibition of action potential firing/neural activity in these cells. These Ai39 mice are useful for optogenetic studies to express an inhibitory opsin that effectively silences the activity of cortical neurons.
Hongkui Zeng, Allen Institute for Brain Science
Genetic Background | Generation |
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F?N3pN1+F21
|
Allele Type | Gene Symbol | Gene Name |
---|---|---|
Targeted (Reporter) | Gt(ROSA)26Sor | gene trap ROSA 26, Philippe Soriano |
Ai39 mice heterozygous for the Rosa-CAG-LSL-eNpHR3.0-EYFP-WPRE conditional allele are viable and fertile. A loxP-flanked STOP cassette prevents transcription of the downstream eNpHR3.0-EYFP fusion gene (see below for detailed description of eNpHR3.0-EYFP). Because the CAG promoter driven reporter construct was targeted for insertion into the Gt(ROSA)26Sor locus, eNpHR3.0-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 eNpHR3.0-EYFP fusion protein. The donating investigator reports that Ai39 mice do not express eNpHR3.0-EYFP prior to introduction of Cre recombinase.
Fusion protein expression following exposure to cre can be detected by EYFP fluorescence and mRNA (in situ hybridization) [and presumably by antibody staining (immunohistochemistry); although this was not tested by the donating investigator]. Following exposure to Cre recombinase, illuminating eNpHR3.0-expressing neurons with yellow-to-red light leads to reversible photoinhibition of action potential firing/neural activity in these cells. The donating investigator specifically reports that expression of the inhibitory opsin occurs at levels sufficient to effectively silence the activity of cortical neurons. Fusion protein expression in tissues other than brain has not yet been evaluated by the donating investigator (April 2011). The phenotype of homozygous mice has not been characterized by the donating investigator. Homozygous mice are viable and fertile.
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.
For characterization information, see images at the Allen Institute for Brain Science website (Ai39 images).
The eNpHR3.0-EYFP fusion protein is composed of the Natronomonas pharaonis-derived halorhodopsin (NpHR) fused in-frame with an enhanced yellow fluorescent protein (EYFP). The eNpHR3.0-EYFP fusion protein has been modified to harbor both the membrane trafficking signal and the endoplasmic reticulum (ER) exporting sequence from the potassium channel Kir2.1 gene. These improvements result in optimized expression in mammalian cells by preventing ER aggregation/enhancing membrane translocation, reducing bleb formation, and enhancing inhibitory capacity. The eNpHR3.0-EYFP fusion protein allows robust photocurrents/action potential inhibition when exposed to light ranging from yellow (~589 nm) to deep red (~660 nm) and at the red/infrared border (~680 nm).
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. The third-generation halorhodopsin eNpHR3.0 is an expression-optimized, yellow-to-red light-driven (~580-680 nm), inward chloride ion pump that causes hyperpolarization and prevents action potentials. For example, illumination of NpHR-expressing neurons leads to reversible photoinhibition of action potential firing/neural activity in these cells.
The Rosa-CAG-LSL-eNpHR3.0-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), an eNpHR3.0-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.
The NpHR-EYFP fusion protein was first designed with the halorhodopsin from the halophilic bacterium Natronomonas pharaonis (NpHR) fused in-frame with an enhanced yellow fluorescent protein (EYFP). To create the eNpHR3.0-EYFP fusion protein, the NpHR-EYFP fusion protein was modified via addition of a membrane trafficking signal from the potassium channel Kir2.1 gene (amino acids KSRITSEGEYIPLDQIDINV) between NpHR and EYFP, as well as addition of an endoplasmic reticulum (ER) exporting sequence from the potassium channel Kir2.1 gene (amino acids FCYENEV) at the C-terminal of the EYFP. These modifications result in optimized expression in mammalian systems by preventing ER aggregation/enhancing membrane translocation, reducing bleb formation, and enhancing inhibitory capacity. The entire Rosa-CAG-LSL-eNpHR3.0-EYFP-WPRE 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 (clone Ai39) were selected.
Chimeric mice were bred with C57BL/6 wildtype mice and/or PhiC31-expressing mice (C57BL/6J congenic background; see Stock No. 007743).
Mutant mice were bred with C57BL/6J wildtype mice for approximately three more generations prior to sending to The Jackson Laboratory Repository. Upon arrival, sperm from Ai39 males was used to fertilize oocytes from C57BL/6J inbred mice (Stock No. 000664) in order to rederive the living colony at The Jackson Laboratory Repository.
All of the mice originally sent to The Jackson Laboratory Repository, as well as all the first generation of rederived offspring, genotype as completely co-segregating for the neo cassette and eNpHR portion of the Ai39 construct. As such, these mice harbor the Ai39 mutation (retaining the attB/attP-flanked PGK-FRT-Neo-polyA cassette) rather than the Ai39Δneo mutation.
As of August 2017, the live colony is ~87-89% C57BL/6.
Expressed Gene | YFP, Yellow Fluorescent Protein, jellyfish |
---|---|
Expressed Gene | HOP, Halorhodopsin, Natronomonas |
Site of Expression | Cre excision of the stop signal results in expression of a halorhodopsin/EYFP (eNpHR3.0-EYFP) fusion protein in cre-expressing tissues. |
Allele Name | targeted mutation 39, Hongkui Zeng |
---|---|
Allele Type | Targeted (Reporter) |
Allele Synonym(s) | Ai39; eNpHR3.0-EYFP; Rosa-CAG-LSL-eNpHR3.0-EYFP |
Gene Symbol and Name | Gt(ROSA)26Sor, gene trap ROSA 26, Philippe Soriano |
Gene Synonym(s) | |
Expressed Gene | YFP, Yellow Fluorescent Protein, jellyfish |
Expressed Gene | HOP, Halorhodopsin, Natronomonas |
Site of Expression | Cre excision of the stop signal results in expression of a halorhodopsin/EYFP (eNpHR3.0-EYFP) fusion protein in cre-expressing tissues. |
Strain of Origin | (129S6/SvEvTac x C57BL/6NCrl)F1 |
Chromosome | 6 |
Molecular Note | The Rosa-CAG-LSL-eNpHR3.0-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), an eNpHR3.0-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. The NpHR-EYFP fusion protein was first designed with the halorhodopsin from the halophilic bacterium Natronomas pharaonis (NpHR) fused in-frame with an enhanced yellow fluorescent protein (EYFP). To create the eNpHR3.0-EYFP fusion protein, the NpHR-EYFP fusion protein was modified via addition of a membrane trafficking signal from the potassium channel Kir2.1 gene (amino acids KSRITSEGEYIPLDQIDINV) between NpHR and EYFP, as well as addition of an endoplasmic reticulum (ER) exporting sequence from the potassium channel Kir2.1 gene (amino acids FCYENEV) at the C-terminal of the EYFP. These modifications result in optimized expression in mammalian systems by preventing ER aggregation/enhancing membrane translocation, reducing bleb formation, and enhancing inhibitory capacity. The entire Rosa-CAG-LSL-eNpHR3.0-EYFP-WPRE targeting vector was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus. |
Mutations Made By | Hongkui Zeng, Allen Institute for Brain Science |
Heterozygous and homozygous mice are viable and fertile. When maintaining a live colony, heterozygous mice may be bred together, to wildtype mice from the colony or to C57BL/6J inbred mice (Stock No. 000664). Alternatively, homozygous mice may be bred together.
When using the Ai39 or Ai39(RCL-eNpHR3.0/EYFP) mouse strain in a publication, please cite the originating article(s) and include JAX stock #014539 in your Materials and Methods section.
Service/Product | Description | Price |
---|---|---|
Heterozygous or Wild-type for Gt(ROSA)26Sor<tm39.1(CAG-HOP/EYFP)Hze> |
Frozen Mouse Embryo | B6;129S-Gt(ROSA)26Sor<tm39(CAG-hop/EYFP)Hze>/J Frozen Embryo | $2595.00 |
Frozen Mouse Embryo | B6;129S-Gt(ROSA)26Sor<tm39(CAG-hop/EYFP)Hze>/J Frozen Embryo | $2595.00 |
Frozen Mouse Embryo | B6;129S-Gt(ROSA)26Sor<tm39(CAG-hop/EYFP)Hze>/J Frozen Embryo | $3373.50 |
Frozen Mouse Embryo | B6;129S-Gt(ROSA)26Sor<tm39(CAG-hop/EYFP)Hze>/J Frozen Embryo | $3373.50 |
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