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.

Figure 1

Generation and analysis of transgenic mouse expressing Cre recombinase in skin epithelia. (A) Schematic representation of transgene, as well as RT-PCR and Northern analysis of Cre mRNA expression in organs taken from our K14–Cre mouse line. RNAs isolated from transgenic organs were subjected to RT-PCR with oligonucleotides specific for β-actin or Cre mRNA. Reverse transcriptase was omitted in control reactions for Cre (No RT Cre). For Northern blot hybridization, Cre cDNA was used as a test probe, and a fragment of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was used as a control. K14Pr, the human K14 promoter; βgint, the 5′ untranslated sequences encompassing an intron from the human β-globin gene; Cre, a full-length cDNA encoding bacterial Cre recombinase; K14pA, 3′ UTR and polyadenylation signal from human K14 mRNA. (B) K14–Cre PCR analysis. DNA was extracted from organs of newborn Cre–MATE/K14–Cre double-positive pups. PCR was carried out with oligonucleotides recognizing either the Cre-driven recombination event (Rec) or the nonrecombined allele (Control). (C) Immunohistochemical assay for Cre activity. The ROSA26 Cre recombinase test allele contains a floxed neo gene separating the ROSA26 promoter (ROSA26 Pr) from the bacterial lacZ gene; under these circumstances, β-galactosidase is expressed only in Cre-active cells that have recombined out the floxed neo gene (31). Tissues from ROSA26 (Left) and from ROSA26/K14–Cre double-positive mice (Right) were stained for β-galactosidase activity. Shown are the results from skins. Triangles, loxP sequences; SA, splice acceptor; neo 4xpA, the neo gene with four polyadenylation signals to ensure transcript termination (31); arrows denote hair follicles. Dotted lines indicate the demarcation between epithelium and mesenchyme. The dark patch to the left of each bar is a microscopy shadow. (Bars = 50 μm.)

Figure 2

Generation and analysis of transgenic mice expressing the CreER^tam fusion protein in skin epithelia. (A) Schematic representation of the transgene. (B) PCR analysis of the DNA extracted from different organs of K14–CreER^tam/Cre–MATE double-positive animals before and after oral administration of tamoxifen. PCR was conducted as described in the legend for Fig. 1. (C) Immunohistochemical assay for Cre activity in transgenic mouse skin. Double-positive K14–CreER^tam/ROSA26 Cre test or single-positive ROSA26 Cre test transgenic mice were used in this experiment. Tamoxifen in DMSO or DMSO alone was applied to the skin once a day for 5 days. A day after the last application, skin biopsies were taken and sections were stained for β-galactosidase activity. Shown are the results from ROSA26 Cre test mouse treated with tamoxifen (Left); double-positive ROSA26 Cre test/K14–CreER^tam mice treated with DMSO (Center); and double-positive mice treated with tamoxifen (Right). Triangles, loxP sequences; the arrow denotes a single cell positive for β-galactosidase in the skin from double-positive ROSA26 Cre test/ K14–CreER^tam mouse treated with DMSO. (Bar = 50 μm.)

Figure 3

Persistence of cells with a Cre-activated, recombined genome: evidence for targeting epidermal stem cells. Double-positive ROSA26 Cre test/K14–CreER^tam transgenic mice were topically treated with tamoxifen once a day for 5 days. Skin biopsies were taken 1 day after the last tamoxifen application (A); 7 days (B); 15 days (C); 30 days (D); 2 months (E); or 4 months (F). Biopsies were sectioned and stained for β-galactosidase activity. The dotted lines denote the demarcation between epithelim and mesenchyme. (Bar = 50 μm.)