Cdh5-Optoα1AR-IRES-lacZ transgenic mice express the light-sensitive effector protein Optoα1AR in vascular endothelial cells (e.g., heart, lung, brain). Light activation of the Optoα1AR molecule in endothelial cells in blood vessels results in increased levels of cytoplasmic IP3 and diacylglycerol (DAG) - which causes a cascade reaction, calcium release, calcium signaling and dilation of overlying smooth muscle.
Michael I Kotlikoff, Cornell University
Cdh5-Optoα1AR-IRES-lacZ transgenic mice express the light-sensitive Optoα1AR fusion protein and lacZ under control of the Cdh5 locus promoter/enhancer regions within the BAC transgene. Cdh5-Optoα1AR-IRES-lacZ mice from founder line B26-1 exhibit high expression levels of Optoα1AR and lacZ in vascular endothelial cells (e.g., heart, lung, brain) - consistent with endogenous Cdh5. Additional details described further below.
The Optoα1AR fusion protein has the transmembrane and light-sensitive domains from bovine G protein-coupled protein rhodopsin with the intracellular domains from human α1a-adrenergic receptor (ADRA1A). When activated with light (~473 nm), Optoα1AR generates the secondary messengers IP3 and diacylglycerol (DAG).
For Cdh5-Optoα1AR-IRES-lacZ transgenic mice, light activation of the Optoα1AR molecule in endothelial cells in blood vessels results in increased levels of cytoplasmic IP3/DAG - which causes a cascade reaction, calcium release, calcium signaling and dilation of overlying smooth muscle.
The donating investigator reports that expression patterns appear to mimic the endogenous Cdh5 expression. Specifically, lacZ staining (after harvesting and staining tissues) was observed in blood vessel endothelial cells of the heart, brain and lung, as well as in vascular endothelial cells of cremaster muscle and femoral artery. They have not tested expression in other tissues to date (March 2019).
Before exposure to the specific wavelength of activating light, lacZ expression is observable (after harvesting and staining tissues), and the Optoα1AR imparts no IP3/DAG release and no endothelial changes. Upon exposure to ~473 nm light, Optoα1AR activation releases IP3/DAG - which causes a cascade reaction, calcium release, calcium signaling and dilation of overlying smooth muscle. Subsequent removal of the activating light returns overlying smooth muscle to the baseline state.
Mice hemizygous for the Cdh5-Optoα1AR-IRES-lacZ transgene are viable and fertile with normal breeding, and no reported gross phenotypic abnormalities. To date (March 2019), it has not been attempted to make this strain homozygous.
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.
Cdh5-Optoα1AR-IRES-lacZ transgenic mice (also called Cdh5BAC-Optoα1AR-IRES-lacZ or Cdh5-Opto-α1AR-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 ~200 kbp C57BL/6J mouse bacterial artificial chromosome (BAC) RP23-453P1 was obtained; containing the entire cadherin 5 locus (Cdh5) as well as ~124 kbp of 5' flanking sequences (including the complete Gm32728, Gm39236 and Gm8748 predicted gene loci) and ~33 kbp of 3' flanking sequences (including a 5' portion of the Bean1 locus). Using homologous recombination/BAC recombineering, a 6170 bp Optoα1AR-IRES-lacZ-pA construct (Optoα1AR, internal ribosome entry site, β-galactosidase and an SV40 polyadenylation signal) was inserted into the ATG start site of the BAC Cdh5 gene (replacing the initiation codon of Cdh5). No other loci on the BAC were altered. The 11.6 kbp BAC vector (pBACe3.6) contained a chloramphenicol selection cassette.
The resulting ~218.1 kbp modified BAC (Cdh5BAC-Optoα1AR-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. Cdh5-Optoα1AR-IRES-lacZ founder line B26-1 was identified with high effector expression in vascular endothelial cells. The donating investigator reports that Cdh5-Optoα1AR-IRES-lacZ transgenic mice from this founder line were backcrossed to C57BL/6J for a total of at least six 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 (March 2019).
The Optoα1AR 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 Gq-coupled human α1a-adrenergic receptor (ADRA1A; α1AR). 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 B26-1, Michael I Kotlikoff|
|Allele Type||Transgenic (Reporter, Inserted expressed sequence, Humanized sequence)|
|Gene Symbol and Name||Tg(Cdh5-RHO/ADRA1,-lacZ)B26-1Mik, transgene insertion B26-1, M I Kotlikoff|
|Strain of Origin||FVB/N X B6(Cg)-Tyrc-2J/J|
|Molecular Note||The ~200 kbp C57BL/6J mouse bacterial artificial chromosome (BAC) RP23-453P1 was obtained; containing the entire cadherin 5 locus (Cdh5) as well as ~124 kbp of 5' flanking sequences (including the complete Gm32728, Gm39236 and Gm8748 predicted gene loci) and ~33 kbp of 3' flanking sequences (including a 5' portion of the Bean1 locus). Using homologous recombination/BAC recombineering, a 6170 bp Optoalpha1AR-IRES-lacZ-pA construct was inserted into the ATG start site of the BAC Cdh5 gene (replacing the initiation codon of Cdh5). No other loci on the BAC were altered. The 11.6 kbp BAC vector (pBACe3.6) contained a chloramphenicol selection cassette. The Optoalpha1AR fusion protein is a G>t<-coupled bovine rhodopsin (RHO) sequence modified to have its intracellular regions (including carboxy-terminal domains) replaced with those from the G>q<-coupled human ADRA1A. Founder line B26-1 was identified with high effector expression in vascular endothelial cells.|
Mice hemizygous for the Cdh5-Optoα1AR-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 (March 2019), it has not been attempted to make this strain homozygous.
When using the Cdh5-Optoα1AR-IRES-lacZ , Cdh5BAC-Optoα1AR-IRES-lacZ , Cdh5-Opto-α1AR-IRES-lacZ mouse strain in a publication, please cite the originating article(s) and include JAX stock #033343 in your Materials and Methods section.
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