The magical touch: Genome targeting in epidermal stem cells induced by tamoxifen application to mouse skin

Abstract

Gene knockout technology has provided a powerful tool for functional analyses of genes expressed preferentially in a particular tissue. Given marked similarities between human and mouse skin, such studies with epidermally expressed genes have often provided valuable insights into human genetic skin disorders. Efficient silencing of a specified gene in a temporally regulated and epidermal-specific fashion could extend functional analyses to broadly expressed genes and increase the categories of human skin disorders to which parallels could be drawn. We have generated transgenic mice expressing Cre and a fusion protein between Cre recombinase and the tamoxifen responsive hormone-binding domain of the estrogen receptor (CreER tam ) under the control of the human keratin 14 (K14) promoter. This promoter is strongly active in dividing cells of epidermis and some other stratified squamous epithelia. With K14-Cre, transgenic embryos recombine genetically introduced loxP sequences efficiently and selectively in the genomes of keratinocytes that reside in embryonic day 14.5 skin, tongue, and esophagus. With K14-CreER tam , postnatal transgenic mice show no Cre activity until tamoxifen is administered. If orally administered, tamoxifen activates keratinocyte-specific CreER tam , allowing recombination of loxP sequences in epidermis, tongue, and esophagus. If topically administered, tamoxifen allows recombination in the area of skin where tamoxifen was applied. Finally, we show that epidermal cells harboring a Cre-dependent rearranged genome persist for many months after tamoxifen application, indicating that the epidermal stem cell population has been targeted efficiently. These tools now pave the way for testing the functional role of different somatic mutations that may exist in mosaic disorders of the skin, including squamous and basal cell carcinomas.

Hair follicle and interfollicular epidermal stem cells make varying contributions to wound regeneration

“…22 To assess the contribution of IFE-derived keratinocytes, we performed lineage tracing studies using mice expressing an inducible Keratin 14 (K14) promoter-driven Cre ERT allele coupled with the YFP reporter (K14;YFP mice). 21 We and others have previously shown that upon tamoxifen treatment, these mice display recombination primarily in the IFE 23,24 (Fig. 1A).…”

“…[19][20][21] These animals were coupled with a Cre-inducible ROSA26R promoter-driven YFP reporter allele, enabling us to genetically label different cell lineages prior to injury. After creating a 0.25 cm 2 full thickness dorsal wound in 7.5 week old telogen skin, we quantitated the number of YFP C cells in the wound up to 50 days post-injury.…”

Basal Cells Are a Multipotent Progenitor Capable of Renewing the Bronchial Epithelium

“…29 To facilitate purification of the fulllength transgene an internal EcoRI site in the original plasmid was eliminated by partial digestion of the plasmid, filling in cohesive ends by Klenow polymerase and ligating the resulting blunt ends. The modified plasmid was digested to yield a 5.25-kb EcoRI/SphI fragment that was microinjected into the male pronucleus of FVB/n zygotes using standard techniques.…”

“…The cell type specificity and ligand-dependence of cremediated recombination was assessed for each K14 line by topical administration of 5 to 10 mg of Tamoxifen (Sigma, St. Louis, MO) in DMSO or DMSO alone to the dorsal skin 29 and by systemic administration of Tamoxifen in corn oil (4 mg Tamoxifen/mouse i.p. on each of 5 consecutive days) to RS and K14/RS bitransgenic.…”

“…Regulation of the recombined allele by the ubiquitous ROSA26 promoter allowed expression of the lineage tag in all cells derived from a K14-expressing progenitor cell. Bitransgenic K14/RS mice derived from each of three K14 lines were screened for ligand-dependent and cell-type-specific recombination in the epidermis following topical administration of vehicle or Tamoxifen 29 and in the bronchial epithelium following systemic administration of vehicle or Tamoxifen using histochemical detection of LacZ on whole mounted tissue. Liganddependent, cell-type-specific recombination was observed in the bronchial epithelium of all 3 lines of K14 transgenic mice.…”