Acta2-Optoβ2AR-IRES-lacZ transgenic mice express the light-sensitive effector protein Optoβ2AR in smooth muscle cells (e.g., blood vessels, lung airways, bladder and gut). Light activation of the Optoβ2AR molecule in smooth muscle results in release of cyclic AMP (cAMP), causing a cascade reaction and muscle relaxation.
Michael I Kotlikoff, Cornell University
Genetic Background | Generation |
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Allele Type |
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Transgenic (Reporter, Inserted expressed sequence) |
Acta2-Optoβ2AR-IRES-lacZ transgenic mice express the light-sensitive Optoβ2AR fusion protein and lacZ under control of the Acta2 locus promoter/enhancer regions within the BAC transgene. Acta2-Optoβ2AR-IRES-lacZ mice from founder line B7-5 exhibit robust expression levels of Optoβ2AR and lacZ in smooth muscle cells (e.g., blood vessels, lung airways, bladder and gut) - consistent with endogenous Acta2. Additional details described further below.
The Optoβ2AR fusion protein has the transmembrane and light-sensitive domains from bovine G protein-coupled protein rhodopsin with the intracellular domains from hamster β2-adrenergic receptor. When activated with light (~473 nm), Optoβ2AR generates the secondary messenger cyclic AMP (cAMP).
For Acta2-Optoβ2AR-IRES-lacZ transgenic mice, light activation of the Optoβ2AR molecule in smooth muscle results in release of cAMP, causing a cascade reaction and muscle relaxation.
The donating investigator reports that expression patterns appear to mimic the endogenous Acta2 expression. Specifically, lacZ staining was observed in blood vessels (heart, lung, diaphragm and brain), small intestine and bladder. Optoβ2AR activation with light was observed in blood vessels (cremaster and femoral vessels). They have not tested expression in other smooth muscle-containing tissues to date (February 2019). Before exposure to the specific wavelength of activating light, lacZ expression is observable, and the Optoβ2AR imparts no cAMP release and no muscle relaxation. Upon exposure to ~473 nm light, Optoβ2AR activation releases cAMP and results in muscle relaxation. Subsequent removal of the activating light returns muscle to the baseline state (allowing contraction).
Mice hemizygous for the Acta2-Optoβ2AR-IRES-lacZ transgene are viable and fertile with normal breeding, and no reported gross phenotypic abnormalities. To date (February 2019), it has not been attempted to make this strain homozygous. As a precaution, the donating investigator does not recommend making this strain homozygous, due to some incidence of lethality in other mouse lines created using the same BAC (e.g., Stock Nos. 025406/028348).
This mouse model is available by way of a collaborative effort between Cornell/National Heart Lung Blood Resource for Optogenetic Mouse Signaling (CHROMus) and The Jackson Laboratory.
Acta2-Optoβ2AR-IRES-lacZ transgenic mice (also called Acta2BAC-Optoβ2AR-IRES-lacZ or Acta2-Opto-β2AR-IRES-lacZ) were designed in the laboratory of Dr. Michael I. Kotlikoff (Cornell University) as part of Cornell/National Heart Lung Blood Resource for Optogenetic Mouse Signaling (CHROMus).
The ~175 kbp C57BL/6J mouse bacterial artificial chromosome (BAC) RP23-370F21 was obtained; containing the entire actin alpha 2 locus (Acta2) as well as ~92 kbp of 5' flanking sequences (including the complete Fas locus) and ~68 kbp of 3' flanking sequences (including the complete Stambpl1 locus). Using homologous recombination/BAC recombineering, a 5990 bp Optoβ2AR-IRES-lacZ-pA construct (Optoβ2AR, internal ribosome entry site, β-galactosidase and an SV40 polyadenylation signal) was inserted into the ATG start site of the BAC Acta2 gene (replacing the initiation codon of Acta2 in exon 2). No other loci on the BAC were altered. The 11.6 kbp BAC vector (pBACe3.6) contained a chloramphenicol selection cassette.
The resulting ~193 kbp modified BAC (Acta2BAC-Optoβ2AR-IRES-lacZ) was purified and then microinjected into the male pronucleus of FVB/N x B6(Cg)-Tyrc-2J/J embryos. Founder animals were bred to C57BL/6J inbred mice for germline transmission. Acta2-Optoβ2AR-IRES-lacZ founder line B7-5 was identified with high sensor expression in smooth muscle. The donating investigator reports that Acta2-Optoβ2AR-IRES-lacZ transgenic mice from this founder line were backcrossed to C57BL/6J for a total of at least ten generations, and then hemizygous males (black coat color) were sent to The Jackson Laboratory Repository in 2019. Upon arrival, sperm was cryopreserved. To establish our living colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).
Of note, the donating investigator reports that, at least once during backcrossing, a hemizygous female was bred to a C57BL/6J inbred male (thus the Y chromosome of the congenic strain is of C57BL/6J origin). The transgene insertion site(s) and transgene copy number were not characterized (February 2019).
The Optoβ2AR fusion protein is a Gt-coupled bovine rhodopsin (RHO) sequence modified to have its intracellular regions (including carboxy-terminal domains) replaced with those from the Gs-coupled hamster β2-adrenergic receptor (Adrb2; β2AR). This fusion protein was designed to be optimized for in vivo expression in mammals. [Airan et al. 2009 Nature 458:1025]
Allele Name | transgene insertion B7-5, Michael I Kotlikoff |
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Allele Type | Transgenic (Reporter, Inserted expressed sequence) |
Allele Synonym(s) | Acta2-Opto beta;2AR-IRES-lacZ |
Gene Symbol and Name | Tg(Acta2-RHO/Adrb2,-lacZ)B7-5Mik, transgene insertion B7-5, M I Kotlikoff |
Gene Synonym(s) | |
Strain of Origin | FVB/N X B6(Cg)-Tyrc-2J/J |
Chromosome | UN |
Molecular Note | The transgenic construct is made using a C57BL/6J mouse bacterial artificial chromosome (BAC) RP23-370F21 containing the entire actin alpha 2 locus (Acta2) as well as ~92 kbp of 5' flanking sequences (including the complete Fas locus) and ~68 kbp of 3' flanking sequences (including the complete Stambpl1 locus). Using homologous recombination/BAC recombineering, a 5990 bp Opto alpha;1AR-IRES-lacZ-pA construct is inserted into the ATG start site of the BAC Acta2 gene (replacing the initiation codon in exon 2). No other loci on the BAC are altered. The 11.6 kbp BAC vector (pBACe3.6) contains a chloramphenicol selection cassette. The Opto beta 2AR fusion protein is a Gt-coupled bovine rhodopsin (RHO) sequence modified to have its intracellular regions (including carboxy-terminal domains) replaced with those from the Gs-coupled hamster Adrb2. Founder line B7-5 has high sensor expression in smooth muscle. |
Mice hemizygous for the Acta2-Optoβ2AR-IRES-lacZ transgene are viable and fertile with normal breeding, and no reported gross phenotypic abnormalities. When maintaining our live colony, hemizygous mice may be bred to wildtype (noncarrier) mice from the colony or to C57BL/6J inbred mice (Stock No. 000664).
To date (February 2019), it has not been attempted to make this strain homozygous. As a precaution, the donating investigator does not recommend making this strain homozygous, due to some incidence of lethality in other mouse lines created using the same BAC (e.g., Stock Nos. 025406/028348).
When using the Acta2-Optoβ2AR-IRES-lacZ , Acta2BAC-Optoβ2AR-IRES-lacZ , Acta2-Opto-β2AR-IRES-lacZ mouse strain in a publication, please cite the originating article(s) and include JAX stock #028347 in your Materials and Methods section.
Facility Barrier Level Descriptions
Service/Product | Description | Price |
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Hemizygous or noncarrier for Tg(Acta2-RHO/Adrb2,-lacZ)B7-5Mik |
Frozen Mouse Embryo | B6.Cg-Tg(Acta2-RHO/Adrb2-lacZ)B7-5Mik/J | $2595.00 |
Frozen Mouse Embryo | B6.Cg-Tg(Acta2-RHO/Adrb2-lacZ)B7-5Mik/J | $2595.00 |
Frozen Mouse Embryo | B6.Cg-Tg(Acta2-RHO/Adrb2-lacZ)B7-5Mik/J | $3373.50 |
Frozen Mouse Embryo | B6.Cg-Tg(Acta2-RHO/Adrb2-lacZ)B7-5Mik/J | $3373.50 |
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