B6;129S-Gt(ROSA)26Sor^{tm34.1(CAG-Syp/tdTomato)Hze}/J

Stock number: 012570
Product name: Ai34D or Ai34(RCL-Syp/tdT)-D
Research areas: Neurobiology Research, Research Tools

Description

Ai34D mice harbor a knock-in mutation of the Gt(ROSA)26Sor locus with a loxP-flanked STOP cassette preventing transcription of a CAG promoter-driven Synaptophysin-tdTomato fusion gene. Following Cre-mediated removal of the STOP cassette, Synaptophysin-tdTomato expression is observed at synaptic termini in brain regions.

Detailed description

Ai34D (or Ai34Δneo) mice heterozygous for the Rosa-CAG-LSL-Synaptophysin-tdTomato-WPRE conditional allele are viable and fertile. A loxP-flanked STOP cassette prevents transcription of the downstream Synaptophysin-tdTomato fusion gene. The Synaptophysin-tdTomato fusion protein is composed of near-full-length mouse synaptophysin protein fused in-frame to the amino terminus of the tdTomato fluorescent protein. Because the CAG promoter driven reporter construct was targeted for insertion into the Gt(ROSA)26Sor locus, Synaptophysin-tdTomato expression is determined by which tissue(s) express Cre recombinase. When bred to mice that express Cre recombinase, the resulting offspring will have the STOP cassette deleted in the cre-expressing tissues; resulting in expression of the Synaptophysin-tdTomato fusion protein. The donating investigator reports that Ai34D mice do not express Synaptophysin-tdTomato prior to introduction of Cre recombinase. Following exposure to Cre recombinase, tdTomato expression can be detected by tdTomato fluorescence and mRNA (in situ hybridization) [and presumably by antibody staining (immunohistochemistry); although this was not tested by the donating investigator]. tdTomato fluorescence is found to be concentrated at synaptic termini in brain regions. Fusion protein expression in tissues other than brain has not yet been evaluated by the donating investigator (April 2011).

Unlike the Ai34 mice from which they were derived, these Ai34D mice no longer harbor the downstream FRT site or attB/attP-flanked selection cassette. The phenotype of homozygous mice has not been characterized by the donating investigator.

For characterization information, see images at the Allen Institute for Brain Science website (Ai34 images).

Development

The Rosa-CAG-LSL-Synaptophysin-tdTomato-WPRE targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), an FRT site, a loxP-flanked STOP cassette (with stop codons in all 3 reading frames and a triple polyA signal), a Synaptophysin-tdTomato fusion protein sequence, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a bovine growth hormone polyA signal, and an attB/attP-flanked PGK-FRT-Neo-polyA cassette.

To create the Synaptophysin-tdTomato fusion protein sequence, a mouse synaptophysin cDNA sequence (encoding a 308 amino acid transcript with all but the first six amino acids of the full-length protein) was fused in-frame via a six nucleotide linker to the amino terminus of the tdTomato sequence (a non-oligomerizing DsRed fluorescent protein variant with a 12 residue linker fusing two copies of the protein (tandem dimer)).

The entire Rosa-CAG-LSL-Synaptophysin-tdTomato-WPRE targeting vector was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus via electroporation of (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells. Correctly targeted ES cells (clone Ai34) were selected. Chimeric mice were bred to PhiC31-expressing mice (C57BL/6J congenic background; see Stock No. 007743) to remove the PGK-FRT-Neo-polyA cassette and replace it with the recombined attB/attP site (attL). The resulting Ai34D (or Ai34Δneo) mice were bred with C57BL/6J wildtype mice for at least two more generations (and the PhiC31 gene was removed) prior to sending to The Jackson Laboratory Repository. Upon arrival, Ai34D mice were bred with C57BL/6J inbred mice (Stock No. 000664) for at least one generations to establish the colony.

Allele

Symbol: Gt(ROSA)26Sor^{tm34.1(CAG-Syp/tdTomato)Hze}
Name: targeted mutation 34.1, Hongkui Zeng
MGI accession ID: MGI:4947243
Generation method: Targeted
Attributes: Reporter
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Chromosome: 6
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: RFP,Red Fluorescent Protein,coral; Syp,synaptophysin,mouse, laboratory
Site of expression: Cre excision of the stop signal results in expression of a Synaptophysin-tdTomato fusion protein in cre-expressing tissues.
Molecular note: The Rosa-CAG-LSL-Synaptophysin-tdTomato-WPRE targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), an FRT site, a loxP-flanked STOP cassette (with stop codons in all 3 reading frames and a triple polyA signal), a Synaptophysin-tdTomato fusion protein sequence, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a bovine growth hormone polyA signal, and an attB/attP-flanked PGK-FRT-Neo-polyA cassette. To create the Synaptophysin-tdTomato fusion protein sequence, a mouse synaptophysin cDNA sequence (encoding a 308 amino acid transcript with all but the first six amino acids of the full-length protein) was fused in-frame via a six nucleotide linker to the amino terminus of the tdTomato sequence (a non-oligomerizing DsRed fluorescent protein variant with a 12 residue linker fusing two copies of the protein (tandem dimer)). PhiC31-mediated recombination removed the neo cassette.