B6.Cg-Tg(Cdh5-RHO/Adrb2,-lacZ)B27-3Mik/J

Stock number: 032889
Product name: Cdh5-Optoβ2AR-IRES-lacZ , Cdh5^BAC-Optoβ2AR-IRES-lacZ , Cdh5-Opto-β2AR-IRES-lacZ
Research areas: Cardiovascular Research, Cell Biology Research, Developmental Biology Research, Internal/Organ Research, Neurobiology Research, Research Tools

Description

Cdh5-Optoβ2AR-IRES-lacZ transgenic mice express the light-sensitive effector protein Optoβ2AR in vascular endothelial cells (e.g., heart, lung, brain). Light activation of the Optoβ2AR molecule in vascular endothelial cells is expected to result in release of cyclic AMP (cAMP), which results in muscle relaxation.

Detailed description

Cdh5-Optoβ2AR-IRES-lacZ transgenic mice express the light-sensitive Optoβ2AR fusion protein and lacZ under control of the Cdh5 locus promoter/enhancer regions within the BAC transgene. Cdh5-Optoβ2AR-IRES-lacZ mice from founder line B27-3 exhibit high expression levels of Optoβ2AR and lacZ in vascular endothelial cells (e.g., heart, lung, brain) - consistent with endogenous Cdh5. 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 Cdh5-Optoβ2AR-IRES-lacZ transgenic mice, light activation of the Optoβ2AR molecule in vascular endothelial cells is expected to result in release of cyclic AMP (cAMP), which results in muscle relaxation.

The donating investigator reports that expression patterns appear to mimic the endogenous Cdh5 expression. Specifically, lacZ staining was observed in endothelial lined capillaries and vessels of the heart, lung and brain. Also, high levels of lacZ expression were observed in diaphragm vasculature. Before exposure to the specific wavelength of activating light, lacZ expression is observable, and the Optoβ2AR imparts no cAMP release. Upon exposure to ~473 nm light, Optoβ2AR activation causes cAMP release and results in muscle relaxation. Subsequent removal of the activating light returns muscle to the baseline state.

Mice hemizygous for the Cdh5-Optoβ2AR-IRES-lacZ transgene are viable and fertile with normal breeding, and no reported gross phenotypic abnormalities. To date (July 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.

Development

Cdh5-Optoβ2AR-IRES-lacZ transgenic mice (also called Cdh5^BAC-Optoβ2AR-IRES-lacZ or Cdh5-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 ~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 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 Cdh5 gene (replacing the initiation codon of Cdh5 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 ~218 kbp modified BAC (Cdh5^BAC-Optoβ2AR-IRES-lacZ) was purified and then microinjected into the male pronucleus of FVB/N x B6(Cg)-Tyr^c-2J/J embryos. Founder animals were bred to C57BL/6J inbred mice for germline transmission. Cdh5-Optoβ2AR-IRES-lacZ founder line B27-3 was identified with high effector and lacZ expression in vascular endothelial cells. The donating investigator reports that Cdh5-Optoβ2AR-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 (July 2019).

The Optoβ2AR 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_s-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

Symbol: Tg(Cdh5-RHO/Adrb2,-lacZ)B27-3Mik
Name: transgene insertion B27-3, Michael I Kotlikoff
MGI accession ID: MGI:6324935
Generation method: Transgenic
Attributes: Reporter; Inserted expressed sequence
Synonyms: B27-Cdh5^BAC-Optobeta 2AR_IRES_lacZ
Marker symbol: Tg(Cdh5-RHO/Adrb2,-lacZ)B27-3Mik
Marker name: transgene insertion B27-3, Michael I Kotlikoff
Chromosome: UN
Strain of origin: FVB/N X B6(Cg)-Tyr^c-2J/J
Molecular note: Cdh5-Optoβ2AR-IRES-lacZ transgenic mice (also called Cdh5-Optoβ2AR-IRES-lacZ or Cdh5-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 ~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 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 Cdh5 gene (replacing the initiation codon of Cdh5 in exon 2).