B6;129S6-Gt(ROSA)26Sor^{tm10(CAG-Syp/EGFP*,-tdTomato)Dym}/J

Stock number: 030206
Product name: RC::FPSit
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

The RC::FPSit dual-recombinase intersectional allele has a frt-flanked STOP and loxP-flanked STOP that prevent transcription of synaptophysin-EGFP fusion protein and tdTomato. Exposure to FLP and Cre recombinase results in strong EGFP expression enriched at the synapse/synaptic vesicle (axonal boutons [terminal and en passant]) of the neuron subtype(s) defined by the overlap of FLP/Cre expression.

Detailed description

The RC::FPSit dual-recombinase intersectional allele has a frt-flanked STOP and loxP-flanked STOP that prevent transcription of two downstream genes - the presynaptic fluorescent fusion protein synaptophysin-EGFP and the tdTomato fluorescent protein. Although under control of the endogenous Gt(ROSA)26Sor promoter/enhancer regions and the CAG hybrid promoter, widespread expression of either fluorescent protein is prevented by STOP cassettes.
Specifically, the donating investigator reports that RC::FPSit mice have virtually no fluorescence prior to introduction of FLP and/or Cre recombinase. After removal of both STOP cassettes by FLP- and Cre-recombination, fluorescent protein expression is observed in those cells where FLP and cre expression overlaps - strong EGFP expression is enriched at the synapse/synaptic vesicle (axonal boutons [terminal and en passant]), whereas weak tdTomato expression is observed. EGFP expression is also observed in the soma and neurites to a certain degree. Furthermore, the tdtomato expression in fixed tissue was only observable by antibody staining and overexposure, and unfixed tissues have not been characterized to date (April 2017). Mice homozygous for the RC::FPSit allele are viable and fertile with no reported breeding abnormalities. These mice are useful for fluorescent visualization of neuron subtype(s) defined by the overlap of FLP/Cre expression, and allow subtype innervation mapping.

Development

The RC::FPSit dual-recombinase intersectional allele was created in the laboratory of Dr. Susan M. Dymecki (Harvard Medical School) to have a CAG promoter, frt-flanked STOP, loxP-flanked STOP and synaptophysin-GFP-IRES-tdTomato all inserted into the Gt(ROSA)26Sor locus. The specific details are below.


The following sequences (from 5' to 3') were subcloned into pRosa26-1: a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), a frt-flanked STOP cassette (containing PGK-neo and lox² transcriptional stop sequences), a loxP-flanked STOP cassette (containing a concatemer of SV40 polyA STOP sequences), a synaptophysin-EGFP fusion gene (described below), an internal ribosome entry site sequence (IRES; allows translation initiation in the middle of an mRNA sequence), a tdTomato gene (cDNA sequence encoding the non-oligomerizing DsRed variant with a 12 residue linker fusing two copies of the protein [tandem dimer]), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), and a rabbit beta-globin polyA sequence. The synaptophysin-EGFP fusion protein (Syp-EGFP) is a full-length mouse synaptophysin (Syp) fused to a enhanced green fluorescent protein.

The final targeting vector (pR26-CAG-FPH-SP-GFP-IRES-tdTomato-WPRE-pA) was inserted into the Gt(ROSA)26Sor locus via electroporation of 129S6/SvEvTac-derived TC-1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts, and chimeras were bred with C57BL/6J mice for germline transmission. The resulting RC::FPSit mice were bred with C57BL/6J wildtype mice for at least one additional generation, and then males with black coat color were sent to The Jackson Laboratory Repository in 2017. Upon arrival, males were used to cryopreserve sperm. To establish the living RC::FrePe mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).
Of note, it is not known if the Y chromosome has been fixed to the C57BL/6J background during backcrossing.

Allele

Symbol: Gt(ROSA)26Sor^{tm10(CAG-Syp/EGFP*,-tdTomato)Dym}
Name: targeted mutation 10, Susan Dymecki
MGI accession ID: MGI:5882396
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; No functional change
Synonyms: RC::FPSit; Rosa26FSF-LSL-Synaptophysin-GFP
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: 129S6/SvEvTac
Expressed genes: GFP,Green Fluorescent Protein,; RFP,Red Fluorescent Protein,coral
Site of expression: After removal of both STOP cassettes by FLP- and Cre-recombination, fluorescent protein expression is observed in those cells where FLP and cre expression overlaps - strong EGFP expression is enriched at the synapse/synaptic vesicle (axonal boutons [terminal and en passant]), whereas weak tdTomato expression is observed. EGFP expression is also observed in the soma and neurites to a certain degree.
Molecular note: The targeting construct includes from 5' to 3' a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter, an FRT-flanked STOP cassette, a loxP-flanked STOP cassette, a synaptophysin-EGFP fusion gene (described below), IRES, a tdTomato gene, a WPRE, and a rabbit beta-globin polyA sequence. The synaptophysin-GFP fusion protein is a full-length mouse synaptophysin gene fused to a mutant enhanced green fluorescent protein (mut4EGFP; harboring the p.V163A/p.S175G codon changes for increased ability to fold properly at 37 degrees C).