D2.Cg-Gt(ROSA)26Sor^{tm1(CAG-EGFP*)Thm}/UgfmJ

Stock number: 038998
Product name: DBA/2J-OMM-GFP
Strain synonyms: MitoTag (Rosa26-CAG-LSL-GFP-OMM) on DBA/2J
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

DBA/2J-OMM-GFP congenic mice were generated through repeated crosses of MitoTag (Rosa26-CAG-LSL-GFP-OMM; Stock No. 032675) animals to inbred DBA/2J mice (Stock No. 000671). The DBA/2J background of this line has made it particularly useful in studies of glaucoma.

MitoTag is a Gt(ROSA)26Sor knock-in allele that has a loxP-flanked STOP cassette preventing transcription of an outer mitochondrial membrane-targeted enhanced green fluorescent protein (GFP-OMM). These mice are designed to allow strong/robust EGFP fluorescence that faithfully localizes to mitochondria (with no observed cytosolic fluorescence) after exposure to Cre recombinase.

Detailed description

DBA/2J-OMM-GFP congenic mice were generated through repeated crosses of MitoTag (Rosa26-CAG-LSL-GFP-OMM; Stock No. 032675) animals to inbred DBA/2J mice (Stock No. 000671). The DBA/2J background of this line has made it particularly useful in studies of glaucoma. The mice can be used to identify retinal ganglion cell (RGC) soma and axons prior to and during glaucomatous degeneration. They can also be used to purify specifically RGC mitochondria from retina or optic nerve via an immuno pull down (PMID 31501572; Fecher, 2019). The iris pigment deposition, increase in intraocular pressure (IOP) and RGC degeneration have not been analyzed in this strain but are expected to match the parent DBA/2J strain. Heterozygotes are viable, fertile, and without overt abnormalities. Homozygous viability/fertility has not been assessed, but is anticipated to be normal (November, 2023).

The original MitoTag allele is a Gt(ROSA)26Sor knock-in allele that has a loxP-flanked STOP cassette preventing transcription of an outer mitochondrial membrane-targeted enhanced green fluorescent protein (GFP-OMM). These mice are designed to allow Cre recombinase-inducible EGFP fluorescence that faithfully localizes to mitochondria (with no observed cytosolic fluorescence).

In an attempt to offer alleles on well-characterized or multiple genetic backgrounds, alleles are frequently moved to a genetic background different from that on which an allele was first characterized. This is the case for the strain above. It should be noted that the phenotype could vary from that originally described. We will modify the strain description, if necessary, as published results become available.

Development

DBA/2J-OMM-GFP congenic mice were generated through backcrosses of MitoTag (Rosa26-CAG-LSL-GFP-OMM; Stock No. 032675) animals to inbred DBA/2J mice (Stock No. 000671 for 13 generations in the laboratory of Dr. Romain Cartoni (then at Duke University). Upon arrival at The Jackson Laboratory, sperm was cryopreserved. Cryorecovery/live colony development will involve DBA/2J (Stock No. 000671).

The original MitoTag allele was created in the laboratory of Dr. Thomas Misgeld (Technical University of Munich) to have a CAG promoter, loxP-flanked STOP cassette and an enhanced green fluorescent protein with an outer mitochondrial membrane tag (GFP-OMM) - all inserted into the Gt(ROSA)26Sor locus. The specific details are below.

The Rosa26-CAG-LSL-GFP-OMM replacement vector was designed with (from 5' to 3') an attB site, a neomycin resistance cassette (with polyA region), a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), a loxP-flanked STOP cassette (LSL; puromycin resistance gene followed by a triple polyA region), the GFP-OMM sequence (described below), a bGH polyA region and an attB site. GFP-OMM is a cDNA sequence composed of an enhanced green fluorescent protein sequence (EGFP) fused at its C-terminal to 37 C-terminal amino acids from the rat Omp25 protein (Synj2bp; aa109-145) which encodes a transmembrane domain for localization to the outer mitochondrial membrane.

(C57BL/6J x 129S6/SvEvTac)F1-derived IDG 3.2 embryonic stem cells previously targeted with a PGK promoter:: attP::hygro-pA::attP in intron 1 of the Gt(ROSA)26Sor locus, were re-transfected with the replacement vector and a phiC31 integrase vector for recombinase-mediated cassette exchange (RCME). Correctly targeted ES cells (Rosa26::PGK::attL::neo::CAG::LSL::GFP-OMM::attL) were injected into recipient blastocysts, and chimeras were bred with C57BL/6NCrl mice for germline transmission. The resulting Rosa26-CAG-LSL-GFP-OMM colony was maintained by backcrossing to C57BL/6NCrl for six generations by the donating laboratory, and then bred together to make homozygous. In 2019, Rosa26-CAG-LSL-GFP-OMM males (black coat color) were sent to The Jackson Laboratory Repository as Stock No. 032675.

Allele

Symbol: Gt(ROSA)26Sor^{tm1(CAG-EGFP*)Thm}
Name: targeted mutation 1, Thomas Misgeld
MGI accession ID: MGI:6296801
Generation method: Targeted
Attributes: Reporter
Synonyms: Gt(ROSA)26Sor^{tm1(CAG-EGFP/Synj2bp)Thm}; MitoTag
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: (C57BL/6J x 129S6/SvEvTac)F1
Molecular note: phiC32-mediated RMCE in ES cells containing PGK promoter::attP::hygro-pA::attP introduced into the loci a construct containing (from 5' to 3') an attB site, a neomycin resistance cassette (with polyA region), a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), a loxP-flanked STOP cassette (LSL; puromycin resistance gene followed by a triple polyA region), a cDNA sequence composed of an enhanced green fluorescent protein sequence (EGFP) fused at its C-terminal to 37 C-terminal amino acids from the rat Omp25 protein (Synj2bp; aa109-145) which encodes a transmembrane domain for localization to the outer mitochondrial membrane, a bGH polyA region, and an attB site.