STOCK Tg(Six2-EGFP/cre)1Amc/J

Stock number: 009606
Research areas: Developmental Biology Research, Research Tools

Description

These Six2-TGC^tg mice express an EGFPCre fusion protein directed to nephron progenitor population cap mesenchyme from the onset of metanephric kidney development by the Six2 (sine oculis-related homeobox 2 homolog (Drosophila)) promoter/enhancer regions within the BAC transgene. Sequence updates published after this transgene was created revealed an upstream Six2 ATG start site that is in-frame with the tTA; thus an apparent and unintended fusion was created with the tTA (80 amino acids potentially added) that renders transgene expression unaffected by tetracycline/doxycycline administration. While not useful as a Tet-Off tool, these Six2-TGC^tg mice may be useful as fluorescent or Cre-lox tools for lineage-tracing/marking Six2-expressing cells for studying multipotent nephron progenitor cell populations throughout kidney organogenesis.

Detailed description

Hemizygous Six2-TGC^tg mice are viable and fertile, harboring a BAC transgene with a Tet-off-eGFPCre under control of the Six2 promoter/enhancer regions within the BAC transgene. The Tet-off-eGFPCre contains both the tetracycline-controlled transactivator protein (tTA) as well as the tetracycline operator (tetO; also called tetracycline-responsive element [TRE] or tet-operator) upstream of an EGFPCre fusion protein. Sequence updates published after this transgene was created revealed an upstream Six2 ATG start site that is in-frame with the tTA; thus an apparent and unintended fusion was created with the tTA (80 amino acids potentially added) that renders transgene expression unaffected by tetracycline/doxycycline administration. It is not known whether the "mutant" tTA is resistant to tetracycline/doxycycline inactivation and still binds to TRE to activate EGFPCre expression or if the TRE acts as a minimal promoter adjacent to the Six2 promoter in the presence of a nonfunctional tTA. Either way, Tet-independent expression of the eGFPCre fusion protein (EGFP immunofluorescence and direct fluorescence/Cre recombinase activity) is directed to nephron progenitor population cap mesenchyme from the onset of metanephric kidney development. When bred with mice containing a loxP-flanked sequence of interest, Cre-mediated recombination will result in deletion of the floxed sequence(s) in such tissues of the offspring. While not useful as a Tet-Off tool, these Six2-TGC^tg mice may be useful as fluorescent or Cre-lox tools for lineage-tracing/marking Six2-expressing cells for studying multipotent nephron progenitor cell populations throughout kidney organogenesis. The donating investigator reports they were unable to produce homozygous mice from hemizygous matings.

Development

The ~181 kb C57BL/6J mouse bacterial artificial chromosome (BAC) RPCI23-311C1, containing the entire Six2 locus (and other genes), was modified by targeting a Tet-off-eGFPCre into the ATG start site of the Six2 locus. This inserted a Tet-off cassette (tetracycline-regulated transactivator [tTA], 2xpolyA signal, tetracycline-responsive element [TRE or tet_o with CMV_min promoter]) followed by an Enhanced Green Fluorescent Protein/Cre Recombinase (EGFP/Cre) fusion protein coding sequence, SV40 polyA signal, and frt-flanked kanamycin cassette. Sequence updates published after this transgene was created revealed an upstream Six2 ATG start site that is in-frame with the tTA; thus an apparent and unintended fusion was created with the tTA (80 amino acids potentially added) that renders transgene expression unaffected by tetracycline/doxycycline administration. The targeted BAC sequences were further modified using FLP recombination to remove the selection cassette and linearized to remove its original vector backbone. The resulting modified BAC was microinjected into CD-1 zygotes. Transgenic founders were obtained and bred to C57BL/6 mice to establish the Six2-TGC^tg colony. These mice were subsequently maintained on CD-1;Swiss Webster;C57BL/6 mixed genetic background prior to arrival at The Jackson Laboratory. Upon arrival, transgenic mice were bred with C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish this colony.

Allele

Symbol: Tg(Six2-EGFP/cre)1Amc
Name: transgene insertion 1, Andrew P McMahon
MGI accession ID: MGI:3845228
Generation method: Transgenic
Attributes: Recombinase-expressing
Marker symbol: Tg(Six2-EGFP/cre)1Amc
Marker name: transgene insertion 1, Andrew P McMahon
Chromosome: UN
Strain of origin: CD-1
Expressed genes: tTA,tetracycline-controlled transactivator,E. coli; GFP,Green Fluorescent Protein,; cre,cre recombinase,bacteriophage P1
Site of expression: EGFP immunofluorescence is observed in nephron progenitor population cap mesenchyme from the onset of metanephric kidney development.
Molecular note: The 181 kb C57BL/6J mouse bacterial artificial chromosome (BAC) RPCI23-311C1, containing the entire Six2 locus (and other genes), was modified by targeting a Tet-off-eGFPCre into the ATG start site of the Six2 locus. This inserted a Tet-off cassette (tetracycline-regulated transactivator (tTA), 2xpolyA signal, tetracycline-responsive element (TRE or tetO with CMV min promoter) followed by an Enhanced Greed Fluorescent Protein/Cre Recombinase (EGFP/Cre) fusion protein coding sequence, (SV40 polyA signal), and frt-flanked kanamycin cassette. The targeted BAC sequences were further modified using FLP recombination to remove the selection cassette and linearized to remove its original vector backbone. The resulting modified BAC was microinjected into CD-1 zygotes. Sequence updates published after this transgene was created revealed an upstream Six2 ATG start site that is in-frame with the tTA; thus an apparent and unintended fusion was created with the tTA (80 amino acids potentially added) that renders transgene expression unaffected by tetracycline/doxycycline administration.