B6.Cg-Tg(Cdh5-COP4*L132C,-lacZ)B28-2Mik/J

Stock number: 033345
Product name: Cdh5-CatCh-IRES-lacZ , Cdh5^BAC-CatCh-IRES-lacZ
Research areas: Cardiovascular Research, Developmental Biology Research, Internal/Organ Research, Neurobiology Research, Research Tools

Description

Cdh5-CatCh-IRES-lacZ transgenic mice express calcium-translocating channelrhodopsin CatCh (an L132C mutant channelrhodopsin with enhanced Ca2+ permeability, accelerated response kinetics and ~70-fold greater light sensitivity) in vascular endothelial cells (e.g., heart, lung, brain). Illumination of the CatCh molecule with blue-to-green light in vascular endothelial cells is expected to result in reversible photostimulation of channel opening, calcium inflow and muscle relaxation.

Detailed description

Cdh5-CatCh-IRES-lacZ transgenic mice express the calcium-translocating channelrhodopsin CatCh and lacZ under control of the Cdh5 locus promoter/enhancer regions within the BAC transgene. Cdh5-CatCh-IRES-lacZ mice from founder line B28-2 exhibit high expression levels of CatCh and lacZ in vascular endothelial cells (e.g., heart, lung, brain) - consistent with endogenous Cdh5. Additional details described further below.

CatCh is a calcium-translocating channelrhodopsin variant designed to have enhanced Ca2+ permeability, accelerated response kinetics and ~70-fold greater sensitivity to blue-to-green light (~450-530 nm; max 474 nm). Additional CatCh information is described below. For Cdh5-CatCh-IRES-lacZ transgenic mice, illumination of the CatCh molecule with this light in vascular endothelial cells is expected to result in reversible photostimulation of channel opening, calcium inflow and relaxation of the muscle.

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. CatCh activation with light has not specifically been tested for Cdh5-CatCh-IRES-lacZ transgenic mice to date (July 2019) - although CatCh function has been shown to work as expected for other CHROMus lines (e.g., Stock Nos. 030334, 033344, etc.).

Mice hemizygous for the Cdh5-CatCh-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-CatCh-IRES-lacZ transgenic mice (also called Cdh5^BAC-CatCh-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 5640 bp CatCh-IRES-lacZ-pA construct (CatCh, 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 ~217.5 kbp modified BAC (Cdh5^BAC-CatCh-IRES-lacZ) was purified and then microinjected into the male pronucleus of C57BL/6J x SJL/J embryos. Founder animals were bred to C57BL/6J inbred mice for germline transmission. Cdh5-CatCh-IRES-lacZ founder line B28-2 was identified with high effector and lacZ expression in vascular endothelial cells. The donating investigator reports that Cdh5-CatCh-IRES-lacZ transgenic mice from this founder line were backcrossed to C57BL/6J for a total of at least five 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 calcium translocating channelrhodopsin CatCh [ChR2(L132C)] cDNA sequence used here encodes the first 309 amino acids of green alga Chlamydomonas reinhardtii channelrhodopsin-2 (ChR2 or cop4) that has been modified with an L132C amino acid substitution (CTC-to-TGC) designed to cause enhanced Ca2+ permeability.

Allele

Symbol: Tg(Cdh5-COP4*L132C,-lacZ)B28-2Mik
Name: transgene insertion B28-2, Michael I Kotlikoff
MGI accession ID: MGI:6324920
Generation method: Transgenic
Attributes: Reporter
Marker symbol: Tg(Cdh5-COP4*L132C,-lacZ)B28-2Mik
Marker name: transgene insertion B28-2, Michael I Kotlikoff
Chromosome: UN
Strain of origin: C57BL/6J x SJL/J
Molecular note: Cdh5-CatCh-IRES-lacZ transgenic mice (also called Cdh5-CatCh-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 5640 bp CatCh-IRES-lacZ-pA construct (CatCh, 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 loc i on the BAC were altered. The 11.6 kbp BAC vector (pBACe3.6) contained a chloramphenicol selection cassette.