C57BL/6J-Tg(Krt8-cre/ERT2,-GFP)1Xin/J

Stock number: 037269
Product name: K8-CreER^T2
Research areas: Cancer Research, Research Tools

Description

K8-creER^T2 transgenic mice express a tamoxifen-inducible Cre recombinase (Cre-ERT2) and a GFP under the control of the Krt8 (keratin 8) promoter. Expression of cre/ERT2 recapitulates Krt8 expression. When bred with mice containing a loxP-flanked sequence, tamoxifen-inducible, cre-mediated recombination will result in deletion of the flanked sequences in Krt8 expressing cells. These mice may be useful in studies of simple epithelial cells, tissue lineage tracing, and in epithelial cancers.

Detailed description

KRT8 (keratin 8 or K8) is a type II keratin that associates with type I keratins (KRT18 or KRT19) to form an intermediate filament in simple single layer epithelial cells. K8-creER^T2 transgenic mice express a tamoxifen-inducible Cre recombinase (Cre-ERT2) and a GFP under the control of the Krt8 promoter. Mice hemizygous for the transgene are viable and fertile. The donating investigator has not generated homozygotes.

K8-creER^T2 transgenic mice express cre in epithelial cells of the bladder, brain, heart, small intestine, kidney, liver, lung, uterus, prostate, skin, thymus, and mammary gland. With the exception of the testis, tamoxifen treated K8-creER^T2 mice express cre and GFP in a pattern similar to endogenous K8. GFP can be detected by PCR and Western blot, however fluorescence is not detected by fluorescent microscopy or FACS. The donating investigator notes that leaky cre activity is observed in the thymus and small intestine. These mice may be useful in studies of simple epithelial cells, tissue lineage tracing, and in epithelial cancers.

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 (17ß-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

BAC clone RP23-234D2 containing the full length mouse Krt8 gene (as well as 60 kb 5' and 100 kb 3' in flanking sequences) was modified to incorporate a cre/ERT2, IRES, GFP, BGH polyA sequence, and a Zeocin resistance gene targeted to exon 1 (downstream of the start codon) of the Krt8 locus. The modified 170 kB BAC was microinjected into the pronucleus of C57BL/6J fertilized oocytes and randomly integrated into the mouse genome. Founder 1 contains two copies of the BAC transgene. The strain was maintained on the C57BL/6J background. Upon arrival, mice were bred to C57BL/6J (Stock No. 000664) for at least one generation to establish the colony.

Allele

Symbol: Tg(Krt8-cre/ERT2,-GFP)1Xin
Name: transgene insertion 1, Li Xin
MGI accession ID: MGI:5314229
Generation method: Transgenic
Attributes: Recombinase-expressing; Reporter; Inducible
Synonyms: K8-CreER^T2; Tg(Krt8-cre/ERT2)1Xin
Marker symbol: Tg(Krt8-cre/ERT2,-GFP)1Xin
Marker name: transgene insertion 1, Li Xin
Chromosome: UN
Strain of origin: C57BL/6J
Molecular note: A BAC clone (RP23-234D2) was modified by recombineering methods to insert the cre/ERT2 fusion sequence at the translational start codon of Krt8. An IRES-GFP expression cassette was inserted downstream of the cre sequence. The linearized BAC construct was microinjected into fertilized C57BL/6J oocytes. RT-PCR was used to screen founder mice and one line was chosen for characterization based on its cre/ERT2 expression pattern which closely recapitulated endogenous Krt8 expression. This line carries 2 copies of the transgene (based on quantitative PCR performed on genomic DNA) with a single insertion site. However, although GFP is detectable by RT-PCR and Western blot analysis, GFP fluorescence cannot be visualized by fluorescent microscopy in tissue sections or by FACS analysis of dissociated prostate cells.