STOCK Tg(tetO-tdTomato,-Syp/EGFP*)1.1Luo/J

Stock number: 012345
Product name: TRE-Bi-SG-T line 1.1
Research areas: Neurobiology Research, Research Tools

Description

These TRE-Bi-SG-T transgenic mice have Myc-tagged tdTomato and synaptophysin/mut4EGFP fusion protein with a bi-directional tetracycline responsive promoter. They are useful to generate mutant mice for Tet-Off/Tet-On and/or fluorescent protein applications, and may be used to design a tripartite system to express fluorescently tagged synaptic proteins with both spatial and temporal control. Expression of tdTomato is directed to the entire cell, while GFP expression is directed to the synapse/synaptic vesicle, allowing labeling of whole neurons in red and presynaptic terminals in green.

Detailed description

Hemizygous TRE-Bi-SG-T line 1.1 transgenic mice are viable and fertile, with no reported phenotypic abnormalities. The TRE-Bi-SG-T transgene has Myc-tagged tdTomato and full-length mouse synaptophysin/mut4EGFP fusion protein (Syp-GFP) expression under the control of the bi-directional tet-responsive promoter (tetO or TRE). When bred with another mouse expressing tetracycline-controlled transactivator protein (tTA) or reverse tetracycline-controlled transactivator protein (rtTA), tdTomato and Syp-GFP fusion protein expression can be regulated with the tetracycline analog doxycycline (dox) in the resulting double mutant offspring. In tTA(-dox) or rtTA(+dox)-expressing cells, tdTomato expression is directed to the entire cell, while GFP expression is directed to the synapse/synaptic vesicle. The donating investigator reports that direct GFP fluorescence and direct tdTomato fluorescence may be visualized in these mice when tTA is present/dox is absent. In addition, the three Myc epitopes at the C-terminus of tdTomato allow fluorescent signal enhancement via immunofluorescence if needed. These TRE-Bi-SG-T transgenic mice are useful to generate mutant mice for Tet-Off/Tet-On and/or fluorescent protein applications.

These TRE-Bi-SG-T transgenic mice may be used to design a tripartite system to express fluorescently tagged synaptic proteins with both spatial and temporal control. When bred with floxed-STOP-tTA mice (ROSA26-ZtTA; Stock No. 012266), the resulting double mutant mice allow tdTomato and Syp-GFP expression to be determined by the tissue-specific Cre recombinase expression of a third strain of the researchers choosing. For example, when TRE-Bi-SG-T transgenic mice are bred with floxed-STOP-tTA mice (ROSA26-ZtTA; Stock No. 012266) and a neural-specific Cre recombinase strain (for example: Foxg1-Cre; Stock Nos. 004337 or 006084), the resulting triple mutant mice should exhibit labeling of whole neurons in red and presynaptic terminals in green in the absence of dox.

Development

The TRE-Bi-SG-T transgene was designed with a tdTomato protein sequence on one side, and a Syp-mut4EGFP fusion protein sequence on the other side of a bi-directional tetracycline-responsive promoter (tetO; also called tetracycline operator, tet-operator, or tetracycline-responsive element [TRE]) with CMV minimal enhancer-less promoter. The tdTomato is tagged with three Myc epitopes at its C-terminus and followed by a polyadenylation signal (tdTomato is a non-oligomerizing DsRed variant with a 12 residue linker fusing two copies of the protein (tandem dimer)). The Syp-mut4EGFP fusion protein is a full-length mouse synaptophysin (Syp) fused to a mutant enhanced green fluorescent protein (mut4EGFP; harboring the V163A/S175G codon changes for increased ability to fold properly at 37 degrees C) and followed by a polyadenylation signal. This TRE-Bi-SG-T transgene was microinjected into the pronuclei of FVB zygotes. Founder mice determined to harbor GFP (via PCR genotyping) were bred to Foxg1-tTA lines on mixed genetic backgrounds (CD1, 129, C57BL/6) to test them for expression and to establish founder lines. Mice from founder line 1.1 were found to have a single copy of the transgene and were bred to animals on mixed genetic backgrounds (mixed CD1, 129, C57BL/6 and/or FVB) for several generations, and then to the outbred CD1 for the last 3 generations prior to sending to The Jackson Laboratory Repository. Upon arrival, mice were bred to C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish the colony.

Allele

Symbol: Tg(tetO-tdTomato,-Syp/EGFP*)1.1Luo
Name: transgene insertion 1.1, Liqun Luo
MGI accession ID: MGI:4461409
Generation method: Transgenic
Attributes: Reporter; Inducible; Inserted expressed sequence
Marker symbol: Tg(tetO-tdTomato,-Syp/EGFP*)1.1Luo
Marker name: transgene insertion 1.1, Liqun Luo
Chromosome: UN
Strain of origin: FVB
Expressed genes: GFP,Green Fluorescent Protein,; RFP,Red Fluorescent Protein,coral; myc tag,myc tag,
Site of expression: When bred to mice expressing a tetracycline transactivator (tTA) or reverse transactivator (rtTA), doxycycline induction results in the expression of tdTomato and synaptophysin/mut4EGFP fusion protein (Syp-GFP) expression in transactivator expressing tissues of the offspring. tdTomato expression is directed to the entire cell, while GFP expression is directed to the synapse/synaptic vesicle.
Molecular note: The TRE-Bi-SG-T transgene was designed with a tdTomato protein sequence on one side, and a Syp-mut4EGFP fusion protein sequence on the other side of a bi-directional tetracycline-responsive promoter (tetO; also called tetracycline operator, tet-operator, or tetracycline-responsive element [TRE]) with CMV minimal enhancer-less promoter. The tdTomato is tagged with three Myc epitopes at its C-terminus and followed by a polyadenylation signal (tdTomato is a non-oligomerizing DsRed variant with a 12 residue linker fusing two copies of the protein (tandem dimer)). The Syp-mut4EGFP fusion protein is a full-length mouse synaptophysin (Syp) fused to a mutant enhanced green fluorescent protein (mut4EGFP; harboring the V163A/S175G codon changes for increased ability to fold properly at 37 degrees C) and followed by a polyadenylation signal.