129S4/SvJae-Gt(ROSA)26Sor^tm8Sor/J

Stock number: 038307
Product name: ROSA26^SAT
Research areas: Cell Biology Research, Neurobiology Research, Research Tools

Description

ROSA26^SAT translocation sensor mice conditionally express fluorescent ERK1/2 pathway, ATK pathway, and nuclear reporters from a single Gt(ROSA)26Sor transcript, enabling live-cell in vivo (whole embryo or animal) and ex vivo (cell line) studies of intracellular signaling in response to external stimuli.

Detailed description

ROSA26^SAT translocation sensor mice conditionally express fluorescent ERK1/2 pathway, ATK pathway, and nuclear reporters from a single Gt(ROSA)26Sor transcript, enabling live-cell in vivo (whole embryo or animal) and ex vivo (cell line) studies of intracellular signaling in response to external stimuli.

An SAT (Splice Acceptor Triple protein) cassette introduced to the mouse Gt(ROSA)26Sor locus incorporates a FOXO1 (forkhead box O1) reporter of the ATK pathway fused to mRuby3, an Extracellular signal-Regulated Kinase-Kinase Translocation Reporter (ERK-KTR) fused to mClover3, and an H2B-mTAGBFP2 (basic blue florescent protein, BFP) nuclear marker. All three reporters are expressed from the same Gt(ROSA)26Sor transcript, separated by a P2A self-cleaving peptides. The ERK-KTR sensor consists of an ERK1/2 binding site and phosphorylation motif adjacent to phosphorylation activated nuclear export signal (NES) and inhibited nuclear localization sequence (NLS) fused to a fluorescent protein (FP). The AKT sensor uses a mutant version of FOXO1 with defective DNA binding and relies on endogenous AKT phosphorylation sites that regulate its nuclear localization. The sensors translocate out of the nucleus in response to signaling. The ERK-KTR and FOXO1 reporters can be deployed simultaneously when tagged with compatible fluorophores, revealing additional information on pathway dynamics and interactions in a single experiment.

Test crosses with mice expressing Wnt1-Cre (H2az2^{Tg(Wnt1-cre)11Rth}, an allelic component of Stock No. 009107) and analysis of mouse embryonic palatal mesenchyme cells (MEPMs) demonstrate that the ERK-KTR reporter in this line behaves similarly to that found in the SAE line (Stock No. 038306, conditionally expressing only the ERK-KTR and nuclear reporters). It shows a nuclear to cytoplasmic shift in localization upon FGF-1 treatment. Some off-target activity by CDK1 has been noted with the ERK-KTR reporter, so this should be considered when interpreting results with this sensor (Wilcockson, 2022). The FOXO1-based AKT reporter behaves as expected, showing an increase in Cyto:Nuc ratio upon growth factor treatment. The donating laboratory notes that it is rare to find cells with FOXO1-mRuby3 predominantly localized to the nucleus, as is often the case for the ERK-KTR reporter. Also of note, it is difficult to image the H2B-mTagBFP2 fusion for an extended period because the MEPMs are very sensitive to the ultraviolet light needed to stimulate the fluorophore.

Development

A viral splice acceptor (SA)-loxP-neomycin-pA-loxP-FOXO1-mRuby3-P2A-H2B-mTagBFP2 (BFP)-P2A-ERK-KTR-mClover3-WPRE-pA cassette was introduced to the mouse Gt(ROSA)26Sor locus through homologous recombination in 129S4/SvJaeSor-derived AK7 embryonic stem (ES) cells. The targeting vector was cloned into the XbaI site in the pROSA26-1 backbone (Addgene, #21714). DNA fragments contained mClover3 (Addgene, #74252), mRuby3 (Addgene, #74252), mTagBFP2 (Addgene, #99267; BFP), human H2B (Addgene, #99267), an adenovirus splice acceptor (Addgene, #21709), a floxed NeoR sequence (Addgene, #13444), the ERK-KTR reporter (Addgene, #59138), the FOXO1 reporter (Addgene, #67759), and woodchuck hepatitis virus post-transcriptional regulatory element (from ROSA26^Tdt mouse genomic DNA; Addgene, #22799). The ERK-KTR sensor consists of an ERK1/2 binding site and phosphorylation motif adjacent to phosphorylation activated nuclear export signal (NES) and inhibited nuclear localization sequence (NLS) fused to a fluorescent protein (FP; mClover3). The AKT sensor uses a mutant version of FOXO1 with defective DNA binding and relies on endogenous AKT phosphorylation sites that regulate its nuclear localization. Resultant mice were backcrossed to 129S4/SvJae mice for at least 2 generations by the donating laboratory.

Upon arrival at The Jackson Laboratory, sperm was cryopreserved. Cryorecovery/live colony development will involve 129S4/SvJaeJ (Stock No. 009104).

Allele

Symbol: Gt(ROSA)26Sor^tm8Sor
Name: targeted mutation 8, Philippe Soriano
MGI accession ID: MGI:7467457
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Synonyms: SAT
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: 129S4/SvJaeSor
Molecular note: A viral splice acceptor (SA)-loxP-neomycin-pA-loxP-FOXO1-mRuby3-P2A-H2B-mTagBFP2 (BFP)-P2A-ERK-KTR-mClover3-WPRE-pA cassette is introduced to the mouse Gt(ROSA)26Sor locus. The targeting vectorincludes DNA fragments contained mClover3 (Addgene, #(a href="https://www.addgene.org/74252/" target="_blank")74252(/a)), mRuby3 (Addgene, #(a href="https://www.addgene.org/74252/" target="_blank")74252(/a)), mTagBFP2 (Addgene, #(a href="https://www.addgene.org/99267/" target="_blank")99267(/a); BFP), human H2B (Addgene, #(a href="https://www.addgene.org/99267/" target="_blank")99267(/a)), an adenovirus splice acceptor, a floxed NeoR sequence, the ERK-KTR reporter, the FOXO1 reporter, and woodchuck hepatitis virus post-transcriptional regulatory element (from ROSA26(Tdt) mouse genomic DNA. The ERK-KTR sensor consists of an ERK1/2 binding site and phosphorylation motif adjacent to phosphorylation activated nuclear export signal (NES) and inhibited nuclear localization sequence (NLS) fused to a fluorescent protein (FP; mClover3). The AKT sensor uses a mutant version of FOXO1 with defective DNA binding and relies on endogenous AKT phosphorylation sites that regulate its nuclear localization.