B6.129S4-Gt(ROSA)26Sor^{tm1(CAG-EGFP/Rpl10a,-birA)Wtp}/J

Stock number: 022367
Product name: Rosa26^fsTRAP
Research areas: Cell Biology Research, Research Tools

Description

fs-TRAP mice are designed to express the EGFP-L10a ribosome tag following exposure to Cre recombinase, and then express 2xHA-tagged BirA (biotin ligase) following exposure to FLP recombinase. This is a tool for the isolation and purification of ribosome-bound proteins and protein complexes from tissues.

Detailed description

These fs-TRAP mice contain from 5' to 3', a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), a floxed-neomycin (neo) resistance cassette and polyadenylation (polyA) sequence, a frt-flanked enhanced green fluorescence protein (EGFP)-L10a (ribosomal protein) fusion protein, a 2xHA (hemagglutinin) tagged bacterial birA, bifunctional protein (biotin ligase), gene, and a polyA sequence. This targeting vector was inserted into the Gt(ROSA)26Sor gene. Mice that are homozygous for the targeted mutation are viable and fertile. Rpl10a encodes the ribosomal protein L10a, an integral component of the 60S ribosomal subunit.

In the absence of Cre, gene expression is prevented by the STOP sequence. After removal of the loxP-flanked neo-polyA cassette via cre-mediated recombination, constitutively activated EGFP-L10a labels ribosomes of selected tissues where Cre recombinase is expressed. Ribosomes of selected tissues are labeled by EGFP, and subsequent immunoprecipitation of EGFP selectively co-precipitates RNAs that actively engaged by ribosomes.

After removal of the frt-flanked EGFP-L10a via FLP-mediated recombination, Cre recombinase expression removes the loxP-flanked neo-polyA cassette, allows for highly specific biotinylation and purification of appropriately tagged proteins.

For example, when crossed to B6.Cg-Tg(Tek-cre)1Ywa/J mice (Stock No. 008863) expressing Cre recombinase in endothelial cells during embryogenesis, this mutant mouse strain may be useful for the isolation of endothelial lineage-selective genes.

In contrast, cBirA mice (B6.129-Gt(ROSA)26Sor^{tm1.1(CAG-birA,EGFP)Joez}/J Stock No. 030420) express BirA upon one cross to Cre Recombinase mice. They do not require an additional cross to Flp Recombinase expressing mice. Also, cBirA mice express EGFP upon one cross to cre-expressing mice.

Development

The fs-TRAP targeting vector was designed with, from 5' to 3', a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), a floxed-neomycin (neo) resistance cassette and polyadenylation (polyA) sequence, a frt-flanked enhanced green fluorescence protein (EGFP)-L10a (ribosomal protein) fusion protein, a 2xHA (hemagglutinin) tagged bacterial birA, bifunctional protein (biotin ligase), gene, and a polyA sequence. This targeting vector was inserted into the Gt(ROSA)26Sor gene. The construct was electroporated into 129S4/SvJae derived J1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. The resulting chimeric animals were crossed to C57BL/6J mice for at least 6 generations by the donating lab (see SNP note below). Upon arrival at The Jackson Laboratory the mice were crossed to C57BL/6J (Stock No. 000664) at least once to establish the colony.

A 48 SNP (single nucleotide polymorphism) panel analysis, with 43 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 43 markers throughout the genome suggested a C57BL/6 genetic background, 3 of 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 mixed C57BL/6J ; C57BL/6N genetic background.

Allele

Symbol: Gt(ROSA)26Sor^{tm1(CAG-EGFP/Rpl10a,-birA)Wtp}
Name: targeted mutation 1, William T Pu
MGI accession ID: MGI:5484611
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: 129S4/SvJae
Expressed genes: GFP,Green Fluorescent Protein,; birA,biotin ligase),bacteria
Site of expression: The EGFP-L10a fusion protein will be expressed in tissues where promoters driving Cre recombinase are expressed.
Molecular note: The fs-TRAP targeting vector was designed with, from 5' to 3', a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), a floxed-neomycin (neo) resistance cassette and polyadenylation (polyA) sequence, a frt-flanked enhanced green fluorescence protein (EGFP)-L10a (ribosomal protein) fusion protein, a 2xHA (hemagglutinin) tagged bacterial birA, bifunctional protein (biotin ligase), gene, and a polyA sequence. This targeting vector was inserted into the Gt(ROSA)26Sor gene.