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.
Michael I Kotlikoff, Cornell University
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 α1a-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.
Lck-Optoα1AR-IRES-lacZ transgenic mice (also called LckBAC-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 (LckBAC-Optoα1AR-IRES-lacZ) was purified and then microinjected into the male pronucleus of FVB/N x B6(Cg)-Tyrc-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 Gt-coupled bovine rhodopsin (RHO) sequence modified to have its intracellular regions (including carboxy-terminal domains) replaced with those from the Gq-coupled human α1a-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 Name||transgene insertion B22-1, M I Kotlikoff|
|Allele Type||Transgenic (Reporter)|
|Allele Synonym(s)||Lck-Opto alpha 1AR-IRES-lacZ|
|Gene Symbol and Name||Tg(Lck-RHO/ADRA1,-lacZ)B22-1Mik, transgene insertion B22-1, M I Kotlikoff|
|Strain of Origin||FVB/N X B6(Cg)-Tyrc-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 Gt-coupled bovine rhodopsin (RHO) sequence modified to have its intracellular regions (including carboxy-terminal domains) replaced with those from the Gq-coupled human alpha 1a-adrenergic receptor.|
Mice hemizygous for the Lck-Optoα1AR-IRES-lacZ transgene are viable and fertile with normal breeding, and no reported gross phenotypic abnormalities. When maintaining our live colony, hemizygous mice may be bred to wildtype (noncarrier) mice from the colony or to C57BL/6J inbred mice (Stock No. 000664).
To date (September 2019), it has not been attempted to make this strain homozygous.
When using the Lck-Optoα1AR-IRES-lacZ , LckBAC-Optoα1AR-IRES-lacZ , Lck-Opto-α1AR-IRES-lacZ mouse strain in a publication, please cite the originating article(s) and include JAX stock #033705 in your Materials and Methods section.
"MICE" means mouse strains, their progeny derived by inbreeding or crossbreeding, unmodified derivatives from mouse strains or their progeny supplied by The Jackson Laboratory ("JACKSON"). "PRODUCT(S)" means biological materials supplied by JACKSON, and their derivatives. "SERVICES" means projects conducted by JACKSON for other parties that may include but are not limited to the use of MICE or PRODUCTS. "RECIPIENT" means each recipient of MICE, PRODUCTS, or SERVICES provided by JACKSON including each institution, its employees and other researchers under its control. MICE or PRODUCTS shall not be: (i) used for any purpose other than internal research, (ii) sold or otherwise provided to any third party for any use, or (iii) provided to any agent or other third party to provide breeding or other services. Acceptance of MICE, PRODUCTS or SERVICES from JACKSON shall be deemed as agreement by RECIPIENT to these conditions, and departure from these conditions requires JACKSON’s prior written authorization.
MICE, PRODUCTS AND SERVICES ARE PROVIDED "AS IS". JACKSON EXTENDS NO WARRANTIES OF ANY KIND, EITHER EXPRESS, IMPLIED, OR STATUTORY, WITH RESPECT TO MICE, PRODUCTS OR SERVICES, INCLUDING ANY IMPLIED WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, OR ANY WARRANTY OF NON-INFRINGEMENT OF ANY PATENT, TRADEMARK, OR OTHER INTELLECTUAL PROPERTY RIGHTS.
In case of dissatisfaction for a valid reason and claimed in writing by a purchaser within ninety (90) days of receipt of, PRODUCTS or SERVICES, JACKSON will, at its option, provide credit or replacement for the PRODUCT received or the SERVICES provided; JACKSON makes no other representations and this shall be the exclusive remedy of the purchaser. Please note specific policy for live mice.
Consistent with the requirement for a written understanding regarding animal care and use, the JACKSON Animal Care and Use Committee will review the animal care and use protocol(s) associated with any SERVICES to be performed at JACKSON, and JACKSON shall have ultimate responsibility and authority for the care of animals while on site or in JACKSON custody.
In no event shall JACKSON, its trustees, directors, officers, employees, and affiliates be liable for any causes of action or damages, including any direct, indirect, special, or consequential damages, arising out of the provision of MICE, PRODUCTS, or SERVICES, including economic damage or injury to property and lost profits, and including any damage arising from acts or negligence on the part of JACKSON, its agents or employees. Unless prohibited by law, in purchasing or receiving MICE, PRODUCTS, or SERVICES from JACKSON, purchaser or recipient, or any party claiming by or through them, expressly releases and discharges JACKSON from all such causes of action or damages, and further agrees to defend and indemnify JACKSON from any costs or damages arising out of any third party claims.
MICE, PRODUCTS or SERVICES are to be used in a safe manner and in accordance with all applicable governmental rules and regulations.
The foregoing represents the General Terms and Conditions applicable to JACKSON’s MICE, PRODUCTS or SERVICES. In addition, special terms and conditions of sale of certain MICE, PRODUCTS, or SERVICES may be set forth separately in JACKSON web pages, catalogs, price lists, contracts, and/or other documents, and these special terms and conditions shall also govern the sale of these MICE, PRODUCTS and SERVICES by JACKSON, and by its licensees and distributors.
Acceptance of delivery of MICE, PRODUCTS or SERVICES shall be deemed agreement to these terms and conditions. No purchase order or other document transmitted by purchaser or recipient that may modify the terms and conditions hereof, shall be in any way binding on JACKSON, and instead the terms and conditions set forth herein, including any special terms and conditions set forth separately, shall govern the sale of MICE, PRODUCTS or SERVICES by JACKSON.