B6.Cg-Tg(Lck-RHO/ADRA1,-lacZ)B22-1Mik/J

Stock number: 033705
Product name: Lck-Optoα1AR-IRES-lacZ , Lck^BAC-Optoα1AR-IRES-lacZ , Lck-Opto-α1AR-IRES-lacZ
Research areas: Cardiovascular Research, Cell Biology Research, Neurobiology Research, Research Tools

Description

Lck-Optoα1AR-IRES-lacZ transgenic mice express the light-sensitive effector protein Optoα1AR in T cells of the spleen. Light activation of the Optoα1AR molecule is expected to generate the secondary messengers IP3 and diacylglycerol (DAG). Lck-Optoα1AR-IRES-lacZ transgenic mice may be useful in studying T cell cytotoxicity, proliferation, and differentiation, as well as InsP3 receptors.

Detailed description

Lck-Optoα1AR-IRES-lacZ transgenic mice express the light-sensitive Optoα1AR fusion protein and lacZ under control of the Lck locus promoter/enhancer regions within the BAC transgene. Lck-Optoα1AR-IRES-lacZ mice from founder line B22-1 exhibit high expression levels of lacZ in T cells of the spleen - consistent with endogenous Lck. 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 Lck-Optoα1AR-IRES-lacZ transgenic mice, the donating investigator reports that expression patterns appear to mimic the endogenous Lck expression. Specifically, lacZ staining was observed in T cell of the spleen (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 Lck-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+ release in T cells regulates cytotoxicity, proliferation, and differentiation in complex ways through highly localized release. InsP3 receptors are a critical component of this control system in ways that are incompletely understood. Lck-Optoα1AR-IRES-lacZ transgenic mice may be useful in studying this.

Mice hemizygous for the Lck-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

Lck-Optoα1AR-IRES-lacZ transgenic mice (also called Lck^BAC-Optoα1AR-IRES-lacZ or Lck-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 ~150 kbp C57BL/6J mouse bacterial artificial chromosome (BAC) RP24-159E19 was obtained; containing the entire lymphocyte protein tyrosine kinase locus (Lck) as well as ~63 kbp of centromeric flanking sequences (including the complete Task3 and Hdac1 loci) and ~77 kbp of telomeric flanking sequences (including the complete Fam167b, Eif3i, Dcdc2b, Iqcc and Ccdc28b loci, as well as a portion of the Txlna 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 Lck gene. No other loci on the BAC were altered. The 13.4 kbp BAC vector contained a chloramphenicol selection cassette.
The resulting ~169.6 kbp modified BAC (Lck^BAC-Optoα1AR-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. Lck-Optoα1AR-IRES-lacZ founder line B22-1 was identified with lacZ expression in the expected cells. The donating investigator reports that Lck-Optoα1AR-IRES-lacZ transgenic mice from this founder line were backcrossed to C57BL/6J for a total of at least seven 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).

In 2020, genotyping performed at The Jackson Laboratory show this Optoα1AR-IRES-lacZ transgene co-segregates with a neomycin resistance gene sequence (suggesting a neo cassette is present within or near this transgene).

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(Lck-RHO/ADRA1,-lacZ)B22-1Mik
Name: transgene insertion B22-1, M I Kotlikoff
MGI accession ID: MGI:6361521
Generation method: Transgenic
Attributes: Reporter
Synonyms: B22-Lck^BAC-Optoa1AR_IRES_lacZ; Lck-Opto alpha 1AR-IRES-lacZ
Marker symbol: Tg(Lck-RHO/ADRA1,-lacZ)B22-1Mik
Marker name: transgene insertion B22-1, M I Kotlikoff
Chromosome: UN
Strain of origin: FVB/N X B6(Cg)-Tyr^c-2J/J
Molecular note: The ~150 kbp C57BL/6J mouse bacterial artificial chromosome (BAC) RP24-159E19 contains the entire lymphocyte protein tyrosine kinase locus as well as ~63 kbp of centromeric flanking sequences (including the complete Task3 and Hdac1 loci) and ~77 kbp of telomeric flanking sequences (including the complete Fam167b, Eif3i, Dcdc2b, Iqcc and Ccdc28b loci, as well as a portion of the Txlna locus). Using homologous recombination/BAC recombineering, a 6170 bp Optoalpha1AR-IRES-lacZ-pA construct is inserted into the ATG start site of the BAC Lck gene. No other loci on the BAC were altered. The 13.4 kbp BAC vector contained a chloramphenicol selection cassette. The 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 alpha 1a-adrenergic receptor.