STOCK Osr1^{tm1(EGFP/cre/ERT2)Amc}/J

Stock number: 009061
Research areas: Cancer Research, Developmental Biology Research, Research Tools

Description

These mice harbor a Osr1^eGFPCreERt2 (Osr1^GCE) "knock-in" allele that both abolishes Osr1 (odd-skipped related 1 (Drosophila)) gene function and expresses EGFP and creERT2 from the Osr1 promoter/enhancer elements. While EGFP expression is observed in intermediate mesoderm and dorsal lateral plate mesoderm of developing embryos (~E8.5), Cre-ERT2 fusion gene activity is inducible; observed in the same cells only following tamoxifen administration. These Osr1^GCE mice may be useful for lineage-tracing/marking Osr1-expressing cells for studying metanephric kidney development, as well as vascular, heart, and urogenital development.

Detailed description

Homozygous mice die upon birth with multiple abnormalities of the heart and urogenital system (among other tissues). Heterozygous mice are viable and fertile. The Osr1^eGFPCreERt2 (Osr1^GCE) "knock-in" allele both abolishes Osr1 gene function and expresses an eGFPCreERt2 fusion protein (EGFP and creERT2 fusion protein from the Osr1 promoter/enhancer elements). While EGFP immunofluorescence is observed in intermediate mesoderm and dorsal lateral plate mesoderm of developing embryos (~E8.5), Cre-ERT2 fusion gene activity is inducible; observed in the same cells only following tamoxifen administration. As such, when Osr1^GCE mice are bred with mice containing loxP-flanked sequence of interest, tamoxifen-inducible, Cre-mediated recombination will result in deletion of the floxed sequences in the Osr1-expressing cells of the offspring. Of note, removal of the frt-flanked neo cassette is reported to result in ectopic Cre recombinase expression in the absence of tamoxifen.

The Cre-ERT2 fusion protein consists of Cre recombinase fused to a triple mutant form of the human estrogen receptor; which does not bind its natural ligand (17β-estradiol) at physiological concentrations but will bind the synthetic estrogen receptor ligands 4-hydroxytamoxifen (OHT or tamoxifen) and, with lesser sensitivity, ICI 182780. Restricted to the cytoplasm, Cre-ERT2 can only gain access to the nuclear compartment after exposure to tamoxifen. To counteract the mixed estrogen agonist effects of tamoxifen injections, which can result in late fetal abortions in pregnant mice, progesterone may be coadministered.

Development

A targeting vector was designed with a cDNA encoding an eGFPCreERt2 (GCE) fusion protein. Specifically, this targeting vector contained an enhanced green fluorescent protein (EGFP) and a CreERT2 fusion gene (Cre-ER^T2; Cre recombinase fused to a G400V/M543A/L544A triple mutation of the human estrogen receptor ligand binding domain), a polyA signal, and a frt-flanked PGK-neo-pA cassette. This construct was designed to replace the first ATG codon of the targeted gene with the first ATG codon of the GCE cassette. This construct was electroporated into F1 hybrid (129/Sv x C57BL/6J) embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts and chimeric mice were bred with C57BL/6J mice. The donating investigator reports that mutant mice were backcrossed to C57BL/6J mice for several generations before arriving at The Jackson Laboratory (see SNP analysis note below). Upon arrival the mice were bred with C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish the colony.

A 27 SNP (single nucleotide polymorphism) panel analysis performed by The Jackson Laboratory revealed 10 of 27 markers (8 different chromosomes) that were not C57BL/6 allele-type. These data indicate the mice were not backcrossed to C57BL/6 prior to arrival at The Jackson Laboratory. No single source of genetic background contamination could be determined by this data. The donating investigator suggests there may be some Swiss Webster genetic contributions from a possible mating with R26Hoxd11 mutant mice many years ago.

Allele

Symbol: Osr1^{tm1(EGFP/cre/ERT2)Amc}
Name: targeted mutation 1, Andrew P McMahon
MGI accession ID: MGI:3827104
Generation method: Targeted
Attributes: Recombinase-expressing; Inducible
Marker symbol: Osr1
Marker name: odd-skipped related transcription factor 1
Chromosome: 12
Strain of origin: Not Specified
Expressed genes: GFP,Green Fluorescent Protein,; cre/ERT2,Cre recombinase and estrogen receptor 1 (human) fusion gene,
Site of expression: GFP immunofluorescence is observed in intermediate mesoderm and dorsal lateral plate mesoderm of developing embryos (~E8.5).
Molecular note: The start codon was replaced with an eGFP/cre/ERT2 cassette followed by an frt flanked neo cassette via homologous recombination. GFP expression was detected in the intermediate mesoderm and dorsal lateral plate mesoderm at E8.5 and E9.5.