129S4/SvJae-Gt(ROSA)26Sor^tm7Sor/J

Stock number: 038306
Product name: ROSA26^SAE
Research areas: Cell Biology Research, Neurobiology Research, Research Tools

Description

ROSA26^SAE translocation sensor mice conditionally express fluorescent ERK1/2 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^SAE translocation sensor mice conditionally express fluorescent ERK1/2 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 SAE (Splice Acceptor ERK) cassette introduced to the mouse Gt(ROSA)26Sor locus incorporates an Extracellular signal-Regulated Kinase-Kinase Translocation Reporter (ERK-KTR) fused to mClover3 and an H2B-mRuby3 nuclear marker. Both are expressed from the same Gt(ROSA)26Sor transcript, separated by a P2A self-cleaving peptide. 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 sensor translocates out of the nucleus in response to signaling.

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 although there is considerable cell-to-cell variability, reporter localization clearly shifts from predominantly nuclear localization during starvation conditions to predominantly cytoplasmic localization following FGF-1 treatment. And although there is variability in the initial state and response to ligand in individual cells, the average response is consistent across experiments (Dinsmore, 2023). 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).

A related conditional strain, ROSA26^SAT (Stock No. 038307), additionally expresses an AKT (AK strain Transforming; protein kinase B, PKB) reporter in order to simultaneously interrogate both the ERK1/2 and AKT pathways.

Development

A viral splice acceptor (SA)-loxP-neomycin-pA-loxP-H2B-mRuby3-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), human H2B (Addgene, #99267), an adenovirus splice acceptor (Addgene, #21709), a floxed NeoR sequence (Addgene, #13444), the ERK-KTR reporter (Addgene, #59138), 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). 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^tm7Sor
Name: targeted mutation 7, Philippe Soriano
MGI accession ID: MGI:7467448
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Synonyms: SAE
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-H2B-mRuby3-P2A-ERK-KTR-mClover3-WPRE-pA cassette is introduced to the mouse Gt(ROSA)26Sor locus. The targeting vector incudes. DNA fragments containing 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)), 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 (Addgene, #(a href="https: //www.addgene.org/59138/" target="_blank")59138(/a)), and woodchuck hepatitis virus post-transcriptional regulatory element. 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).