C57BL/6-Gt(ROSA)26Sor^{tm7(Pik3ca*,EGFP)Rsky}/J

Stock number: 012343
Product name: R26Stop^FLP110*
Research areas: Cancer Research, Cell Biology Research, Immunology, Inflammation and Autoimmunity Research, Research Tools

Description

The R26Stop^FLP110* conditional allele is targeted to the Gt(ROSA)26Sor locus and has a loxP-flanked Neo-STOP cassette preventing transcription of the phosphatidylinositol 3-kinase, catalytic, alpha polypeptide gene (Pik3ca or P110α) and EGFP. These R26Stop^FLP110* mice allow inducible expression of activated PIK3 heterodimer activity and may be useful in studying signaling cascades that control cell survival, growth, and proliferation, as well as differentiation, regeneration, hypertension and cancer.

Detailed description

Mice homozygous for the R26Stop^FLP110* conditional allele (also called P110*-transgene) are viable and fertile, with a loxP-flanked Neo-STOP cassette preventing transcription of the downstream bicistronic sequences (P110* [a constitutively active form of the mouse catalytic P110α subunit of phosphatidylinositol 3-kinase] and EGFP). When bred to mice that express Cre recombinase, offspring will have the STOP cassette deleted and subsequent expression of the P110* signal molecule and EGFP fluorescence in the cre-expressing cells. Expression of P110* leads to constitutively active PIK3 heterodimer activity; resulting in the generation of downstream effectors that mediate signal transduction cascades that control cell survival and cell cycle progression (growth, and proliferation). Of note, breeding these mice to an FLP-expressing strain will result in removal of the frt-flanked IRES-EGFP cassette.

Development

The P110*-transgene is a targeting vector designed with (from 5' to 3') a splice acceptor site, a loxP-flanked Neo-STOP cassette, a cDNA sequence encoding the constitutively active P110*, an frt-flanked internal ribosomal entry site-enhanced green fluorescent protein cassette (IRES-EGFP), and bovine polyadenylation signal. P110* is a mutant form of mouse P110α (Pik3ca; the catalytic subunit of phosphatidylinositol 3-kinase) that was made constitutively active by addition of the p85 iSH2 domain (region between the two Src homology 2 domains of p85 that is required for the enzymatic activity of p110) fused to the p110 N terminus using a flexible glycine-kinker hinge region. This transgene construct was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus via electroporation into C57BL/6-derived embryonic stem (ES) cells. Correctly targeted ES cells were selected and chimeric animals were bred to C57BL/6 to generate the mutant colony. These R26Stop^FLP110* mice were maintained on a C57BL/6 genetic background (see SNP note below) as a homozygous colony for many generations prior to arrival at The Jackson Laboratory Repository. Upon arrival, mutant mice were bred to C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish the colony.

A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. While the 27 markers throughout the genome suggested a C57BL/6 genetic background, all 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a C57BL/6N genetic background.

Allele

Symbol: Gt(ROSA)26Sor^{tm7(Pik3ca*,EGFP)Rsky}
Name: targeted mutation 7, Klaus Rajewsky
MGI accession ID: MGI:4430527
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Constitutively active; Inserted expressed sequence
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Chromosome: 6
Strain of origin: C57BL/6
Expressed genes: GFP,Green Fluorescent Protein,
Site of expression: Cre excision of the stop signal results in expression of EGFP and a constitutively active form of P110 in cre-expressing tissues.
Molecular note: The P110*-transgene is a targeting vector designed with (from 5' to 3') a splice acceptor site, a loxP-flanked Neo-STOP cassette, a cDNA sequence encoding P110* (a constituitively active form of mouse P110alpha [Pik3ca; the catalytic subunit of phosphatidylinositol 3-kinase]), an FRT-flanked internal ribosomal entry site-enhanced green fluorescent protein cassette (IRES-EGFP), and bovine polyadenylation signal. This transgene construct was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus.