B6.Cg-Gt(ROSA)26Sor^{tm3(CAG-EYFP)Hze}/J

Stock number: 007903
Product name: Ai3 or Ai3(RCL-EYFP)
Research areas: Neurobiology Research, Research Tools

Description

These Ai3 mice harbor a targeted mutation of the Gt(ROSA)26Sor locus with a loxP-flanked STOP cassette preventing transcription of a CAG promoter-driven enhanced yellow fluorescent protein (EYFP), and may be useful as a Cre reporter strain. EYFP is expressed following Cre-mediated recombination.

Detailed description

Ai3 mice hemizygous for this Rosa-CAG-LSL-EYFP-WPRE conditional allele are viable and fertile. A loxP-flanked STOP cassette prevents transcription of the downstream enhanced yellow fluorescent protein (EYFP). When bred to mice that express Cre recombinase, the resulting offspring will have the STOP cassette deleted in the cre-expressing tissue(s); resulting in expression of EYFP. Because this CAG promoter driven reporter construct was targeted for insertion into the Gt(ROSA)26Sor locus, EYFP expression is determined by which tissue(s) express Cre recombinase. The donating investigator reports that Ai3 mice do not express EYFP prior to introduction of Cre recombinase and EYFP expression following exposure to cre can be detected by fluorescence, mRNA (in situ hybridization) and antibody staining (immunohistochemistry). These Ai3 mice are useful as a Cre reporter strain; expressing enhanced yellow fluorescent protein following Cre-mediated recombination.

For characterization information, see images at the Allen Institute for Brain Science website (Ai3 images).

Of note, the FRT sites flanking the mutation allow for additional targeted replacement of the reporter sequences through Flp-mediated recombination if so desired.

Similarly, the attB/attP-flanked selection cassette may be removed by introduction of the site-specific bacteriophage PhiC31 integrase if so desired.

Development

The Rosa-CAG-LSL-EYFP-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), an enhanced yellow fluorescent protein sequence (from pcDNA 6.2/N-YFP-DEST (Invitrogen)), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a polyA signal, and an attB/attP-flanked PGK-FRT-Neo-polyA cassette. This entire construct 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 Ai3) were selected and chimeric males were bred to C57BL/6J females. The resulting Ai3 mice were then backcrossed to C57BL/6J for at least 3 generations prior to arrival at The Jackson Laboratory. Upon arrival, mice were further backcrossed to C57BL/6J inbred mice (Stock No. 000664) to generate this congenic colony.

Allele

Symbol: Gt(ROSA)26Sor^{tm3(CAG-EYFP)Hze}
Name: targeted mutation 3, Hongkui Zeng
MGI accession ID: MGI:3809521
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); 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: YFP,Yellow Fluorescent Protein,jellyfish
Site of expression: A floxed STOP prevents transcription of EYFP until mice are bred to a Cre strain. Resulting offspring express EYFP in a pattern dictated by the Cre promoter.
Molecular note: The Rosa-CAG-LSL-EYFP-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), an enhanced yellow fluorescent protein sequence (from pcDNA6.2/N-YFP-DEST), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a polyA signal, and an attB/attP-flanked PGK-FRT-Neo-polyA cassette. This entire construct was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus.