B6.Cg-Gt(ROSA)26Sor^{tm6(CAG-ZsGreen1)Hze}/J

Stock number: 007906
Product name: Ai6 or Ai6(RCL-ZsGreen)
Research areas: Neurobiology Research, Research Tools

Description

Ai6 is a Cre reporter allele designed to have a loxP-flanked STOP cassette preventing transcription of a CAG promoter-driven enhanced green fluorescent protein variant (ZsGreen1) - all inserted into the Gt(ROSA)26Sor locus. Ai6 mice express robust ZsGreen1 fluorescence following Cre-mediated recombination.

Detailed description

Ai6 mice hemizygous for this Rosa-CAG-LSL-ZsGreen1-WPRE conditional allele are viable and fertile. A loxP-flanked STOP cassette is designed to prevent high transcription levels of the downstream enhanced green fluorescent protein variant ZsGreen1 (see below). 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 high expression of ZsGreen1. Because this CAG promoter-driven reporter construct is inserted into the Gt(ROSA)26Sor locus, ZsGreen1 expression is determined by which tissue(s) express Cre recombinase. These Ai6 mice are useful as a Cre reporter strain; expressing the enhanced green fluorescent protein, ZsGreen1, following Cre-mediated recombination.



Importantly, some ZsGreen1 expression may be present prior to introduction of Cre recombinase - but the ZsGreen1 expression levels after Cre recombination are significantly greater than those baseline levels. As such, it is recommended that researchers include Cre-negative Ai6 controls to establish the baseline ZsGreen1 levels in their experiments.



The donating investigator reports ZsGreen1 expression following Cre recombinase exposure can be detected by fluorescence and mRNA (in situ hybridization). Bright fluorescence is observed mainly in cell bodies.



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.

For characterization information, see images at the Allen Institute for Brain Science website (Ai6 images).

Development

The Rosa-CAG-LSL-ZsGreen1-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), ZsGreen1 sequence (from pZsGreen1-N1 (Clontech); a human codon optimized/enhanced green fluorescent protein engineered for brighter fluorescence and higher expression in mammalian cells), 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 Ai6) were selected and chimeric males were bred to Cre-transgenic females (expressing cre in non-germ cells from mixed genetic backgrounds). The resulting Ai6 mice (still retaining the floxed-STOP cassette and not harboring the Cre transgene) were then backcrossed to C57BL/6J for at least 1 generation 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^{tm6(CAG-ZsGreen1)Hze}
Name: targeted mutation 6, Hongkui Zeng
MGI accession ID: MGI:3809522
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: GFP,Green Fluorescent Protein,
Site of expression: A floxed STOP prevents transcription of enhanced green fluorescent protein (ZsGreen1) until mice are bred to a Cre strain. Resulting offspring express ZsGreen1 in a pattern dictated by the Cre promoter.
Molecular note: The Rosa-CAG-LSL-ZsGreen1-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), ZsGreen1 sequence (from pZsGreen1-N1 (Clontech); a human codon optimized/enhanced green fluorescent protein engineered for brighter fluorescence and higher expression in mammalian cells), 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.