The Six2CE knock-in/knock-out allele was designed to both abolish expression of the Six2 gene and direct expression of the CreERT2 fusion protein from the Six2 promoter/enhancer elements. These mice are a Cre-lox tool that allows inducible Cre expression in Six2-expressing cells/tissues; including developing nephron progenitor cells.
Andrew P McMahon, University of Southern California
The Six2, sine oculis-related homeobox 2, gene encodes a transcription factor that is important in embryonic development, in particular, the maintenance of kidney nephron progenitors. Six2 is expressed in metanephric mesenchymal stem cells during kidney development and is not expressed in adult kidneys. In this inducible knock-in/knock-out Six2CE strain, Cre-ERT2 protein expression is driven by the endogenous Six2 promoter (a CreERT2 cassette replaced the initiation codon of Six2). Expression of the targeted Six2 is ablated and Cre activity is only detected upon treatment with tamoxifen, thereby offering both spatial and temporal control. Tamoxifen-inducible cre activity is observed in the developing kidney, head, ear and limb, mimicking the endogenous Six2 expression pattern.
Upon crossing these mice to Cre-reporter strains (e.g. floxed lacZ or GFP), Six2 expressing cells may be labeled for lineage tracing.
Heterozygous mice are viable and fertile. Homozygotes are not viable, dying shortly after birth.
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.
A targeting construct was designed to insert a CreERT2 fusion gene (Cre-ERT2; Cre recombinase fused to a G400V/M543A/L544A triple mutation of the human estrogen receptor ligand binding domain), and a FRT-PGKneobpA-FRT into the first ATG codon of the targeted Six2 gene. This construct was inserted into the targeted gene via electroporation into (C57BL/6 x 129S4/SvJae)F1-derived v6.5 embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6J blastocysts. The resulting chimeric animals were tested for germline transmission. The mice were backcrossed to the C57BL/6J background since 2007. During backcrossing, the Y chromosome may not have been fixed to the C57BL/6J genetic background.
Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).
|Allele Name||targeted mutation 2, Andrew P McMahon|
|Allele Type||Targeted (Recombinase-expressing, Inducible)|
|Allele Synonym(s)||Six2CE; Six2CreERT2; Six2-CE; Six2-CreERT2; Six2-CreERT|
|Gene Symbol and Name||Six2, sine oculis-related homeobox 2|
|Strain of Origin||(C57BL/6 x 129S4/SvJae)F1|
|Molecular Note||A targeting vector was designed to insert a CreERT2 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.|
When maintaining a live colony, heterozygous mice may be bred to wildtype siblings, or to C57BL/6J inbred mice (Stock No. 000664). Homozygotes are not viable.
When using the Six2CE, Six2-CE mouse strain in a publication, please cite the originating article(s) and include JAX stock #032488 in your Materials and Methods section.