B6;SJL-Tg(Dbh-COP4*L132C,-lacZ)B16-4Mik/J

Stock number: 032666
Product name: Dbh-CatCh-IRES-lacZ , Dbh^BAC-CatCh-IRES-lacZ
Research areas: Cardiovascular Research, Cell Biology Research, Endocrine Deficiency Research, Internal/Organ Research, Neurobiology Research, Research Tools

Description

Dbh-CatCh-IRES-lacZ transgenic mice express the calcium-translocating channelrhodopsin CatCh (an L132C mutant channelrhodopsin with enhanced Ca2+ permeability, accelerated response kinetics and ~70-fold greater light sensitivity) in sympathetic neurons in brain and chromaffin cells of the adrenal medulla. Illumination of the CatCh molecule with blue-to-green light in neurons is expected to result in reversible photostimulation of channel opening, calcium flow and depolarization/activation of the cells.

Detailed description

Dbh-CatCh-IRES-lacZ transgenic mice express the calcium-translocating channelrhodopsin CatCh and lacZ under control of the Dbh locus promoter/enhancer regions within the BAC transgene. Dbh-CatCh-IRES-lacZ mice from founder line B16-4 exhibit high expression levels of lacZ in sympathetic neurons in brain and chromaffin cells of the adrenal medulla - consistent with endogenous Dbh. 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 Dbh-CatCh-IRES-lacZ transgenic mice, illumination of the CatCh molecule with this light in neurons is expected to result in reversible photostimulation of channel opening, calcium flow and depolarization/activation of the cells.

The donating investigator reports that expression patterns appear to mimic the endogenous Dbh expression. Specifically, lacZ staining was observed in sympathetic neurons in brain and chromaffin cells of the adrenal medulla (after harvesting and staining tissues [either whole organ or by sectioning and then staining]). They have not tested expression in other tissues to date (September 2019).
CatCh activation with light has not specifically been tested for Dbh-CatCh-IRES-lacZ transgenic mice to date (September 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 Dbh-CatCh-IRES-lacZ transgene are viable and fertile with normal breeding, and no reported gross phenotypic abnormalities. To date (September 2019), it has not been attempted to make this strain homozygous.

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. Chlamydomonas reinhardtii-derived channelrhodopsin-2 (ChR2 or cop4) is a blue light-driven cation channel that depolarizes the cell and causes action potentials. The calcium translocating channelrhodopsin (CatCh) is a ChR2 variant modified to have an L132C amino acid substitution for enhanced Ca2+ permeability; resulting in accelerated response time and a voltage response that is ~70-fold more light sensitive than wildtype ChR2. As such, illuminating CatCh-expressing cells with blue-to-green light (~450-530 nm; max 474 nm) leads to rapid and reversible photostimulation of channel opening (e.g, calcium inflow and contraction of muscle, or robust action potential firing activity in neurons, etc.). Because CatCh activation is possible at naturally occurring light intensities while maintaining high temporal precision, it may also have applications in gene-therapeutic visual restoration efforts.

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

Dbh-CatCh-IRES-lacZ transgenic mice (also called Dbh^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 ~188.5 kbp C57BL/6J mouse bacterial artificial chromosome (BAC) RP23-354N13 was obtained; containing the entire dopamine beta-hydroxylase locus (Dbh), as well as ~106 kbp centromeric flanking sequences (including the complete Adamtsl2 locus) and ~65 kbp telomeric flanking sequences (including the complete Sardh and Sardhos loci). 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 Dbh gene. No other loci on the BAC were altered. The 11.6 kbp BAC vector (pBACe3.6) contained a chloramphenicol selection cassette.
The resulting ~205.7 kbp modified BAC (Dbh^BAC-CatCh-IRES-lacZ) was purified and then microinjected into the male pronucleus of (C57BL/6J x SJL/J)F2 embryos. Founder animals were bred to C57BL/6J inbred mice for germline transmission. Dbh-CatCh-IRES-lacZ founder line B16-4 was identified with high lacZ expression in the expected cells. The donating investigator reports that Dbh-CatCh-IRES-lacZ transgenic mice from this founder line were backcrossed to C57BL/6J for a total of five generations (see SNP results below). In 2019, hemizygous males (black coat color) were sent to The Jackson Laboratory Repository. 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 (September 2019).



In 2020, a 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the males sent to The Jackson Laboratory Repository. This revealed 1-3 of 43 markers were not fixed for C57BL/6 allele-type (i.e., still segregating for SJL allele-type markers [3 different chromosomes]). These data suggest that the mice sent to The Jackson Laboratory Repository were incompletely backcrossed onto C57BL/6 (~93% C57BL/6).

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(Dbh-COP4*L132C,-lacZ)B16-4Mik
Name: transgene insertion B16-4, M I Kotlikoff
MGI accession ID: MGI:6361518
Generation method: Transgenic
Attributes: Reporter
Marker symbol: Tg(Dbh-COP4*L132C,-lacZ)B16-4Mik
Marker name: transgene insertion B16-4, M I Kotlikoff
Chromosome: UN
Strain of origin: (C57BL/6J x SJL/J)F2
Molecular note: The ~188.5 kbp C57BL/6J mouse bacterial artificial chromosome (BAC) RP23-354N13 was obtained; containing the entire dopamine beta-hydroxylase locus (Dbh), as well as ~106 kbp centromeric flanking sequences (including the complete Adamtsl2 locus) and ~65 kbp telomeric flanking sequences (including the complete Sardh and Sardhos loci). Using homologous recombination/BAC recombineering, a 5640 bp CatCh-IRES-lacZ-pA construct was inserted into the ATG start site of the BAC Dbh gene. No other loci on the BAC were altered. The 11.6 kbp BAC vector (pBA Ce3.6) contained a chloramphenicol selection cassette. Founder line B16-4 was identified with high lacZ expression in the expected cells. The calcium translocating channelrhodopsin cDNA sequence used here encodes the first 309 amino acids of green alga Chlamydomonas reinhardtii channelrhodopsin-2 that has been modified with an L132C amino acid substitution (CTC-to-TGC) designed to cause enhanced Ca2+ permeability.