B6;129-Six2^{tm3(EGFP/cre/ERT2)Amc}/J

Stock number: 009600
Product name: Six2^GCE , Six2-GCE
Research areas: Cancer Research, Developmental Biology Research, Research Tools

Description

This Six2^GCE knock-in/knock-out allele was designed to both abolish Six2 gene function and express EGFP and creERT2 from the Six2 promoter/enhancer elements. While EGFP expression is observed in nephron progenitor population cap mesenchyme from the onset of metanephric kidney development, Cre-ERT2 fusion gene activity is inducible observed in the same cells only following tamoxifen administration. These Six2^GCE mice may be useful for lineage-tracing/marking Six2-expressing cells for studying multipotent nephron progenitor cell populations throughout kidney organogenesis.

The donating investigator has also made available a Six2 knock-in/knock-out allele expressing CreERT2 (Six2^CE ; Stock No. 032488) and a BAC transgenic expressing an EGFP/Cre fusion protein (Six2-TGC^tg ; Stock No. 009606).

Detailed description

While heterozygous mice are viable and fertile with no reported abnormalities, homozygous mice die shortly after birth. The Six2^GCE "knock-in" allele both abolishes Six2 gene function and expresses an eGFPCreER^T2 fusion protein (EGFP and creERT2 fusion protein) from the Six2 promoter/enhancer elements. While EGFP immunofluorescence is observed in nephron progenitor population cap mesenchyme from the onset of metanephric kidney development, Cre-ERT2 fusion gene activity is inducible; observed in the same cells only following tamoxifen administration. As such, when Six2^GCE mice are bred with mice containing loxP-flanked sequence, tamoxifen-inducible, Cre-mediated recombination will result in deletion of the floxed sequences in the Six2-expressing cells of the offspring.

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 to insert an eGFPCreER^T2 (GCE) fusion protein coding sequence and frt-flanked PGKneobpA selection cassette into the initiation codon of the Six2 (sine oculis-related homeobox 2 homolog (Drosophila)) targeted gene. Specifically, this targeting vector contained an enhanced green fluorescent protein (EGFP) and a CreER^T2 fusion gene (Cre-ER^T2; Cre recombinase fused to a G400V/M543A/L544A triple mutation of the human estrogen receptor ligand binding domain), an SV40 polyA signal, and an frt-flanked PGK-neo-bpA 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 males were bred with C57BL/6J females. These Six2-GCE (Six2^GCE) mutant mice were maintained on this mixed C57BL/6J;129/Sv genetic background prior to arrival at The Jackson Laboratory. Upon arrival, mice were bred with C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish the colony.

Allele

Symbol: Six2^{tm3(EGFP/cre/ERT2)Amc}
Name: targeted mutation 3, Andrew P McMahon
MGI accession ID: MGI:3845222
Generation method: Targeted
Attributes: Recombinase-expressing; Reporter; Inducible; Null/Knockout
Synonyms: Six2^eGFPCreERT2; Six2^GCE; Six2-eGFP-cre^Tg; Six2-eGFPCreER^T2; Six2-eGFPCreERT2; Six2-GCE
Marker symbol: Six2
Marker name: sine oculis-related homeobox 2
Chromosome: 17
Strain of origin: (129/Sv x C57BL/6J)F1
Expressed genes: GFP,Green Fluorescent Protein,; cre/ERT2,Cre recombinase and estrogen receptor 1 (human) fusion gene,
Site of expression: EGFP immunofluorescence is observed in nephron progenitor population cap mesenchyme from the onset of metanephric kidney development.
Molecular note: The targeting vector was designed to insert an eGFPCreERT2 (GCE) fusion protein coding sequence and frt-flanked PGKneobpA selection cassette into the initiation codon of the Six2 (sine oculis-related homeobox 2 homolog (Drosophila)) targeted gene.