Rosa26CAG-STOP-Foxn1-IRES-GFP knock-in mice are useful in studies of thymopoiesis, and as a source of reprogrammed thymic epithelial cells in vitro culture, or for thymic grafts in vivo.
Nancy Manley, University of Georgia
The targeted Foxn1 gene encodes a transcription factor that is essential for thymic epithelium development. Mutations in this gene have been associated with T-cell immunodeficiency, and Alopecia. These mice carry a floxed STOP cassette, upstream of Foxn1 cDNA, inserted into the Gt(ROSA)26Sor locus. Cre recombinase-mediated excision of the floxed STOP permits site-directed selective control of Foxn1 and GFP expression. Mice that are homozygous for the targeted mutation are viable and fertile.
When crossed to an inducible Cre/ERT2 expressing strain, tamoxifen treatment results in Foxn1 expression levels similar to that observed in fetal thymic epithelial cells. MEFs from the double mutant progeny (iFoxn1) can be sorted and isolated by flow cytometry using GFP expression, and can be used in transplantation studies to generate a functional thymus in vivo.
A targeting vector containing (from 5’ to 3’) a CAG promoter, loxP site, FRT flanked neo cassette, stop cassette, loxP site, Foxn1 cDNA, IRES, GFP, polyadenylation signal sequence was inserted into the Gt(ROSA)26Sor locus. The construct was electroporated into 129P2/OlaHsd derived E14tg2a embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6 blastocysts. The resulting chimeric animals were tested for germline transmission. The mice were bred to Tg(CAG-FLPe) mice to remove the FRT site flanked neo selection cassette. The mice were then backcrossed to C57BL/6J for 5 generations. 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 2.1, C Clare Blackburn|
|Allele Type||Targeted (Conditional ready (e.g. floxed), Reporter, Inserted expressed sequence)|
|Allele Synonym(s)||iFoxn1; Rosa26CAG-STOP-Foxn1-IRES-GFP|
|Gene Symbol and Name||Gt(ROSA)26Sor, gene trap ROSA 26, Philippe Soriano|
|Strain of Origin||129P2/OlaHsd|
|Molecular Note||A construct containing a CAG promoter, loxP site, FRT flanked neo cassette, stop cassette, loxP site, Foxn1 cDNA, IRES and GFP gene was inserted in the locus via homologous recombination. Flp mediated recombination removed the neo cassette.|
When maintaining a live colony, homozygous mice may be bred together.
When using the Rosa26CAG-STOP-Foxn1-IRES-GFP mouse strain in a publication, please cite the originating article(s) and include JAX stock #020625 in your Materials and Methods section.