B6N.Cg-Gt(ROSA)26Sor^{tm1(CAG-EGFP*)Thm}/J

Stock number: 032675
Product name: MitoTag (Rosa26-CAG-LSL-GFP-OMM)
Strain synonyms: B6N.Cg-Gt(ROSA)26Sor^{tm1(CAG-EGFP/Synj2bp)Thm}/J
Research areas: Cancer Research, Cell Biology Research, Metabolism Research, Neurobiology Research, Research Tools

Description

MitoTag (Rosa26-CAG-LSL-GFP-OMM) 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.

This mutation is also available on a DBA/2J genetic background - see Stock No. 038998.

Detailed description

MitoTag (Rosa26-CAG-LSL-GFP-OMM) 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).

Prior to Cre recombinase exposure, the donating investigator reports the floxed-STOP cassette prevents expression of GFP-OMM. Both heterozygous and homozygous mice are viable and fertile with normal breeding, and no reported gross physical or behavioral abnormalities.

Following Cre recombinase exposure, deletion of the STOP cassette allows GFP-OMM to be expressed - resulting in outer mitochondrial membrane-tagged EGFP fluorescence that can be robust and bright. Specifically, the donating investigator reports GFP-OMM faithfully localizes to mitochondria (with no observed cytosolic fluorescence) in all Cre recombinase-expressing lines they tested, including GFAP-Cre line 77.6 (astrocytes; Stock No. 024098), L7-Cre (Purkinje cells; Stock No. 004146), Emx1-Cre (forebrain neural cells; Stock No. 005628), ChAT-Cre (cholinergic neurons; Stock No. 006410), Cx3cr1^CreER (monocytes/macrophages; Stock No. 020940), LysMcre (myeloid cell lineage; Stock No. 004781), PLP/creERT (oligodendrocytes/Schwann cells; Stock No. 005975), Pvalb-T2A-Cre-D (scattered interneuron populations; Stock No. 012358), TRPV1^Cre (dorsal root ganglia, peripheral sensory nerve endings and CNS; Stock No. 017769), Sst-IRES-Cre (dendritic inhibitory interneurons; Stock No. 018973), Vip-IRES-cre (GABAergic interneurons; Stock No. 010908) and CAGGCre-ER^TM (widespread cells; Stock No. 004682).
In addition, double mutant ChAT-Cre;GFP-OMM mice exhibited no adverse effects on mitochondrial or cellular health in motor neurons (i.e., normal mitochondrial transport and shape, as well as neuromuscular synapse morphology).

The Jackson Laboratory Repository has several lines with Cre recombinase-inducible mitochondrial fluorescence, including Dr. Thomas Misgeld's MitoTag (Rosa26-CAG-LSL-GFP-OMM) (Stock No. 032675 ; Rosa26::PGK::att::neo::CAG::loxP-STOP-loxP::GFP-OMM::att), Dr. David Sabatini's MITO-Tag (Stock No. 032290 ; Rosa26::CAG::loxP-STOP-loxP::3xHA-EGFP-SYNJ2BP::lacZ::BGH-pA) and Dr. Jeremy Nathans' ROSA-MitoGFP (Stock No. 021429 ; Rosa26::CAG::loxP-STOP-loxP::MitoGFP-3xV5::BGH::frt-PGK-neo-frt). While both the MitoTag (Rosa26-CAG-LSL-GFP-OMM) and MITO-Tag constructs utilize an EGFP sequence fused at its C-terminal to 37 C-terminal amino acids from the rat Omp25 protein (Synj2bp ; aa109-145), the ROSA-MitoGFP construct utilizes a GFP sequence fused at its N-terminal to the mitochondrial-targeting sequence of cytochrome c oxidase VIIIa (C8a) and fused at its C-terminal to triple V5 epitope. As such, in comparison to ROSA-MitoGFP, the GFP-OMM is smaller in size - which may aid in its faithful localization to mitochondria with no observed cytosolic fluorescence (see detailed characterization above). In addition, Dr. Misgeld reports GFP-OMM can be robust and bright, but may be less bright when directly compared to ROSA-MitoGFP due to differences in fluorophore accommodation between mitochondrial matrix and outer mitochondrial membrane.

Development

The MitoTag (Rosa26-CAG-LSL-GFP-OMM) 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::att::neo::CAG::LSL::GFP-OMM::att [see additional note below]) 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. Upon arrival, males were used to cryopreserve sperm. To establish our living Rosa26-CAG-LSL-GFP-OMM colony in 2019, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6NJ inbred females (Stock No. 005304).

Of note, it is not known if the Y chromosome has been fixed to the C57BL/6 background during backcrossing.

In 2021, 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 rederived living colony at The Jackson Laboratory Repository. One of the 43 markers throughout the genome, on Chromosome 18, was segregating with 129. Also, some of the mice born in this colony are black and tan, suggesting a spontaneous recessive coat color mutation may be present.

Additional Note regarding the att sites: This MitoTag allele is Rosa26::PGK::att::neo::CAG::LSL::GFP-OMM::att, but it has not been determined if the att sites present are Rosa26::PGK::attL::neo::CAG::LSL::GFP-OMM::attR or Rosa26::PGK::attR::neo::CAG::LSL::GFP-OMM::attL [September 2026].

Allele

Symbol: Gt(ROSA)26Sor^{tm1(CAG-EGFP*)Thm}
Name: targeted mutation 1, Thomas Misgeld
MGI accession ID: MGI:6296801
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); 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.