C57BL/6J-Igs2^{tm2(CAG-NanoLuc/CyOFP1)Mzlin}/J

Stock number: 038889
Product name: CAG-LSL-KZ-Antares
Research areas: Neurobiology Research, Research Tools

Description

CAG-LSL-KZ-CyOFP1-NanoLuc-CyOFP1 mice harbor the Antares conditional allele, a highly catalytic blue-emitting modified firefly luciferase (NanoLuc), fused to two copies of the cyan excitable fluorescent protein, CyOFP1, that with its' substrates, hydrofurimazine, cephalofurimazine, and fluorofurimazine provide improved solubility and higher brightness. This strain may be useful for inducible tissue specific bioluminescence imaging and bioluminescent resonance energy transfer (BRET).

Of note, strains for bioluminescent imaging from the Lin Laboratory (Stanford) include Akaluc (Stock No. 038888), Antares (Stock No. 038889), CaMB1 300 (Stock No. 038890), CaMB1 110 (Stock No. 038891) and Fluc2 (Stock No. 038892).

Detailed description

Antares conditional knockin mice harbor the CAG-LSL-KZ-CyOFP1-NanoLuc-CyOFP1 fusion bioluminescent reporter (Antares), designed with a CAG promoter, loxP-STOP-loxP and an Antares coding sequence inserted into the H11 (Igs2) locus. Antares is a highly catalytic blue-emitting modified firefly luciferase (NanoLuc) derived from Oplophorus gracilis and fused to two copies of the cyan excitable fluorescent protein CyOFP1. Used in bioluminescence imaging (BLI), Antares can be used with improved analogs of its substrate furimazine as a bright bioluminescent reporter. In the liver, the substrate hydrofurimazine has enhanced aqueous solubility allowing delivery of higher doses. A second substrate, fluorofurimazine, provides higher brightness in vivo. And a third substrate, cephalofurimazine, provides 20 fold more signal in the brain than luciferase/luciferan. Prior to recombinase exposure, this allele is designed to have a STOP cassette preventing expression of Antares. When bred to mice expressing Cre recombinase, offspring will have the STOP cassette deleted in the cells where cre is expressed resulting in expression of Antares.

Of note, strains for bioluminescent imaging from the Lin Laboratory (Stanford) include Akaluc (Stock No. 038888), Antares (Stock No. 038889), CaMB1 300 (Stock No. 038890), CaMB1 110 (Stock No. 038891) and Fluc2 (Stock No. 038892).

Development

The Antares conditional knockin allele was created using site-specific PhiC31 integrase-mediated transgenesis to insert a loxP-flanked stop cassette, a Kozak sequence and the CyOFP1-NanoLuc-CyOFP1 fusion bioluminescent reporter (Antares) directed by a chicken beta-actin promoter with a CMV enhancer (CAG promoter), inserted into the intergenic site 2 locus (Igs2, also called H11) on chromosome 11. Antares is composed of the highly-catalytic, blue-emitting firefly luciferase derivative NanoLuc fused to two copies of the fluorescent protein CyOFP1. CyOFP1 is a basic (constitutively fluorescent) long stokes shift fluorescent protein derived from Entacmaea quadricolor. The construct was microinjected, along with the ΦC31 (PhiC31) integrase mRNA and dDNA (donor plasmid DNA coded Antares flanked by two attB sites) into the pronuclei of zygotes collected from C57BL/6J mice, which were then implanted into recipient pseudo-pregnant females. Subsequent progeny were screened by PCR for the presence of the Antares allele. Offspring were bred with C57BL/6J mice to confirm germline transmissibility. The resulting Antares mice were then backcrossed to C57BL/6J mice for one generation , male and female heterozygous offspring were mated to get homozygous Antares mice. The homozygous Antares male and female mice were kept breeding for 5 generatiosn before males were sent to The Jackson Laboratory in 2023. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Allele

Symbol: Igs2^{tm2(CAG-NanoLuc/CyOFP1)Mzlin}
Name: targeted mutation 2, MIchael Z Lin
MGI accession ID: MGI:7532826
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Synonyms: H11P-CAG-LSL-Antares
Marker symbol: Igs2
Marker name: intergenic site 2
Marker synonyms: H11; Hipp11; Iis2
Chromosome: 11
Strain of origin: C57BL/6J
Molecular note: The allele is created using site-specific PhiC31 integrase-mediated transgenesis to insert a loxP-flanked stop cassette, a Kozak sequence and the CyOFP1-NanoLuc-CyOFP1 fusion bioluminescent reporter (Antares) directed by a chicken beta-actin promoter with a CMV enhancer (CAG promoter). Antares is composed of the highly-catalytic, blue-emitting firefly luciferase derivative NanoLuc fused to two copies of the fluorescent protein CyOFP1. CyOFP1 is a basic (constitutively fluorescent) long stokes shift fluorescent protein derived from Entacmaea quadricolor.