B6.Cg-Tg(Aldh1l1-RHO/ADRA1,-lacZ)B36-3Mik/J

Stock number: 033706
Product name: Aldh1L1-Optoα1AR-IRES-lacZ , Aldh1L1^BAC-Optoα1AR-IRES-lacZ , Aldh1L1-Opto-α1AR-IRES-lacZ
Research areas: Cardiovascular Research, Cell Biology Research, Neurobiology Research, Research Tools

Description

Aldh1L1-Optoα1AR-IRES-lacZ transgenic mice express the light-sensitive effector protein Optoα1AR in astrocytes in the brain. Light activation of the Optoα1AR molecule is expected to generate the secondary messengers IP3 and diacylglycerol (DAG). Aldh1L1-Optoα1AR-IRES-lacZ transgenic mice may be useful in studying Ca2+ signaling in astrocytes that results in the release of transmitters that modulate neuronal and vascular function, as well as InsP3 receptors.

Detailed description

Aldh1L1-Optoα1AR-IRES-lacZ transgenic mice express the light-sensitive Optoα1AR fusion protein and lacZ under control of the Aldh1l1 locus promoter/enhancer regions within the BAC transgene. Aldh1L1-Optoα1AR-IRES-lacZ mice from founder line B36-3 exhibit high expression levels of lacZ in astrocytes - consistent with endogenous Aldh1L1. 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 α_1a-adrenergic receptor (ADRA1A). When activated with light (~473 nm), Optoα1AR generates the secondary messengers IP3 and diacylglycerol (DAG).

For Aldh1L1-Optoα1AR-IRES-lacZ transgenic mice, the donating investigator reports that expression patterns appear to mimic the endogenous Aldh1l1 expression. Specifically, lacZ staining was observed in astrocytes in the brain (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).
Optoα1AR activation with light has not specifically been tested for Aldh1L1-Optoα1AR-IRES-lacZ transgenic mice to date (September 2019) - although Optoα1AR function has been shown to work as expected for another CHROMus line (e.g., Stock No. 033343).
Ca2+ signaling in astrocytes results in the release of transmitters that modulate neuronal and vascular function. This signaling may occur in fine astrocytes processes that rely on local Ca2+ release from InsP3 receptors. Aldh1L1-Optoα1AR-IRES-lacZ transgenic mice may be useful in studying this.

Mice hemizygous for the Aldh1L1-Optoα1AR-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.

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

Aldh1L1-Optoα1AR-IRES-lacZ transgenic mice (also called Aldh1L1^BAC-Optoα1AR-IRES-lacZ or Aldh1L1-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 ~228 kbp C57BL/6J mouse bacterial artificial chromosome (BAC) RP23-7M9 was obtained; containing the protein coding region of the aldehyde dehydrogenase 1 family, member L1 locus (Aldh1l1) as well as ~170 kbp of telomeric flanking sequences (including the complete Slc41a3 locus and a portion of the Iqsec1 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 Aldh1l1 gene. No other loci on the BAC were altered. The 11.6 kbp BAC vector contained a chloramphenicol selection cassette.
The resulting ~245.8 kbp modified BAC (Aldh1L1^BAC-Optoα1AR-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. Aldh1L1-Optoα1AR-IRES-lacZ founder line B36-3 was identified with lacZ expression in the expected cells. The donating investigator reports that Aldh1L1-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 (September 2019).

The Optoα1AR 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 α_1a-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

Symbol: Tg(Aldh1l1-RHO/ADRA1,-lacZ)B36-3Mik
Name: transgene insertion B36-3, M I Kotlikoff
MGI accession ID: MGI:6361524
Generation method: Transgenic
Attributes: Reporter
Marker symbol: Tg(Aldh1l1-RHO/ADRA1,-lacZ)B36-3Mik
Marker name: transgene insertion B36-3, M I Kotlikoff
Chromosome: UN
Strain of origin: (C57BL/6J x SJL/J)F2
Molecular note: The ~228 kbp C57BL/6J mouse bacterial artificial chromosome (BAC) RP23-7M9 was obtained; containing the protein coding region of the Aldh1l1 as well as ~170 kbp of telomeric flanking sequences (including the complete Slc41a3 locus and a portion of the Iqsec1 locus). Using homologous recombination/BAC recombineering, a 6170 bp Opto alpha1AR-IRES-lacZ-pA construct is inserted into the ATG start site of the BAC Aldh1l1 gene. No other loci on the BAC were altered. The 11.6 kbp BAC vector contained a chloramphenicol selection cassette. The fusion protein is a Gt-coupled bovine rhodopsin sequence modified to have its intracellular regions (including carboxy-terminal domains) replaced with those from the Gq-coupled human ADRA1A.