STOCK Igs2^{tm4(CAG-CaMBI110)Mzlin}/J

Stock number: 038891
Product name: CAG-LSL-CaMBI-110
Research areas: Neurobiology Research, Research Tools

Description

CAG-LSL-CaMBI-110 mice harbor the CamBI 110 conditional allele, Antares-derived, orange-emitting, calcium-modulated bioluminescent indicator that with its' substrate, hydrofurimazine provides 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

Orange CamBI 110 conditional knockin mice harbor the CAG-LSL-KZ-CamBI 110 bioluminescent reporter designed with a CAG promoter, loxP-STOP-loxP and an CamBI 110 coding sequence inserted into the H11 (Igs2) locus. CamBI 110 is an Antares-derived, orange-emitting, calcium-modulated bioluminescent indicator with dissociation constant (Kd) for calcium at 110 nM. Used in bioluminescence imaging (BLI), CamBI 110 can be used with improved analogs of its substrate furimazine as a bright bioluminescent reporter. In the liver, the sustained release substrate hydrofurimazine provides orange signals bright enough to be seen by the naked eye for greater than an hour. Prior to recombinase exposure, this allele is designed to have a STOP cassette preventing expression of CamBI 110. 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 CamBI 110.

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 Orange CamBI 110 conditional knockin allele was created using site-specific PhiC31 integrase-mediated transgenesis to insert a loxP-flanked stop cassette, and the Antares-derived, orange-emitting, calcium-modulated bioluminescent indicator (CaMBI) followed by a polyA signal and directed by a chicken beta-actin promoter with a CMV enhancer (CAG promoter) followed by a polyA signal and inserted into the intergenic site 2 locus (Igs2, also called H11) on chromosome 11. CamBI 110 is composed of CyOFP1::N-terminal NLuc::1aa linker (I)::CaM::4aa linker (GGGS)::M13::2aa linker (EL)::C-terminal NLuc::CyOFP1. The CaM-Glinker-M13 is inserted at position 133 of NanLuc domain to confer responsivity to calcium with maximal brightness. The construct was microinjected, along with the ΦC31 (PhiC31) integrase mRNA and dDNA (donor plasmid DNA coded CamBI 94 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 CamBI 110 allele. Offspring were bred with C57BL/6J mice to confirm germline transmissibility. The resulting CamBI 110 mice (F0, CamBI94 het) were then backcrossed to Albumin-cre mice (B6.Cg-Speer6-ps1^{Tg(Alb-cre)21Mgn}/J, Stock 003574) for one additional generation; male and female offspring (F1) were mated to get homozygous CambBI 110 mice (F2). The homozygous CamBI 110 male and female mice were kept breeding for 8 generations by the donating laboratory (see SNP note below). 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).

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. Two of the 43 markers throughout the genome were segregating with an unknown source, on Chromosomes 1 and 5, while 5 markers were segregating with DBA. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed genetic background.

Allele

Symbol: Igs2^{tm4(CAG-CaMBI110)Mzlin}
Name: targeted mutation 4, Michael Z Lin
MGI accession ID: MGI:7532790
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Synonyms: CAG-LSL-CaMBI-110; Orange CaMBI 110
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, and the Antares-derived, orange-emitting, calcium-modulated bioluminescent indicator (CaMBI) followed by a polyA signal and directed by a chicken beta-actin promoter with a CMV enhancer (CAG promoter). CamBI 110 is composed of CyOFP1::N-terminal NLuc::1aa linker (I)::CaM::4aa linker (GGGS)::M13::2aa linker (EL)::C-terminal NLuc::CyOFP1.