B6N.129S6(Cg)-Krt5^{tm1.1(cre/ERT2)Blh}/J

Stock number: 029155
Product name: K5-CreER^T2 knock-in
Research areas: Cancer Research, Research Tools

Description

Krt5-CreERT2 knock-in mice have both endogenous gene and tamoxifen-inducible Cre recombinase expression directed to basal epithelial cells by the endogenous promoter/enhancer elements of the keratin 5 locus. These mice may be useful in generating conditional alleles for studying basal cells, cancer and epidermolysis bullosa.

Of note, the same Krt5-CreERT2 knock-in allele is also available on the FVB/N genetic background as Stock No. 036976.

Detailed description

The keratin 5 gene (Krt5) encodes cytokeratin-5 or K5, a type II cytokeratin specifically expressed in basal epithelial cells (including epidermis, salivary gland and digestive tract). K5 is a biomarker for several different types of cancer (including breast and lung).

The Krt5-CreERT2 knock-in allele has an IRES sequence and a tamoxifen-inducible Cre recombinase (CreER^T2) inserted in the 3' UTR of Krt5. As such, Krt5-CreERT2 mice have both endogenous gene and CreER^T2 expression directed to basal epithelial cells by the endogenous promoter/enhancer elements of the keratin 5 locus. CreER^T2 fusion gene activity is inducible; observed following tamoxifen administration. When Krt5-CreERT2 mice are bred with mice containing loxP-flanked sequences, tamoxifen-inducible Cre-mediated recombination will result in deletion of floxed sequences in the Krt5-expressing cells of the double mutant offspring.

Specifically, tamoxifen-inducible Cre recombinase activity recapitulates the endogenous Krt5 expression pattern. Recombination efficiency is reported to be high in the basal layer of the epidermis, prostate and esophagus, but only 25% in the basal cells of the trachea (which may reflect the lower Krt5 expression in this tissue). The donating investigator did not characterize if Cre recombinase is expressed prior to tamoxifen exposure.

Mice heterozygous for this Krt5-CreERT2 knock-in allele are viable and fertile with normal breeding, and no reported gross phenotypic or behavioral abnormalities. The donating investigator has not attempted to make this C57BL/6N Krt5-CreERT2 knock-in strain homozygous to date (May 2016), however the same allele on FVB/N is reported to not produce homozygotes (see Stock No. 036976). The Jackson Laboratory has received information that a researcher bred C57BL/6N Krt5-CreERT2 knock-in mice together and the resulting homozygotes developed dermatitis as early as 5 weeks of age that was severe by 11-16 weeks of age [September 2022].

When Krt5-CreERT2 mice are bred to have a cre-conditional knockout of suppressor of cytokine signals 3 (Socs3^flox; Stock No. 010944) and fluorescent cre-reporter (R26-LSL-EYFP; Stock No. 006148), the resulting triple mutant mice allow tamoxifen-inducible deletion of Socs3 in basal cells and activation of YFP expression as a lineage tracer. Such animals are useful for studying negative feedback regulation / inhibition of the JAK/STAT3 pathway.

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

The Krt5-CreERT2 knock-in allele (K5-CreER, K5-CreER^T2 or K5CREER) was created by the laboratory of Dr. Brigid LM Hogan (Duke University Medical Center) to have an IRES-CreER^T2 inserted into the 3' UTR of the keratin 5 gene (Krt5) on chromosome 15. The specific details are below.

The targeting vector contained, from 5' to 3', an internal ribosome entry site (IRES), 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) and an frt-flanked neomycin selection cassette. This construct was electroporated into 129S6/SvEvTac-derived embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts and chimeric males were bred with B6;SJL-Tg(ACTFLPe)9205Dym/J females (Stock No. 003800) to remove the neo selection cassette. The resulting Krt5-CreERT2 colony was maintained by breeding to C57BL/6NCrl wildtype mice for at least eight generations (and the FLPe transgene was removed) prior to sending male mice to The Jackson Laboratory Repository in 2016. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6N oocytes (Stock No. 005304).

Allele

Symbol: Krt5^{tm1.1(cre/ERT2)Blh}
Name: targeted mutation 1.1, Brigid L Hogan
MGI accession ID: MGI:5305438
Generation method: Targeted
Attributes: Recombinase-expressing; Inducible
Marker symbol: Krt5
Marker name: keratin 5
Chromosome: 15
Strain of origin: 129S6/SvEvTac
Expressed genes: cre/ERT2,Cre recombinase and estrogen receptor 1 (human) fusion gene,
Site of expression: Tamoxifen-inducible Cre recombinase is expressed in basal epithelial cells.
Molecular note: An IRES-cre/ERT2 fragment was inserted into the 3'UTR of the Krt5 gene along with an FRT flanked neomycin selection cassette. The selection cassette was subsequenty excised from properly targeted mice using flp recombinase.