Spatio-temporally controlled site-specific somatic mutagenesis in the mouse - PubMed (original) (raw)

Spatio-temporally controlled site-specific somatic mutagenesis in the mouse

J Brocard et al. Proc Natl Acad Sci U S A. 1997.

Abstract

The efficient introduction of somatic mutations in a given gene, at a given time, in a specific cell type will facilitate studies of gene function and the generation of animal models for human diseases. We have shown previously that conditional recombination-excision between two loxP sites can be achieved in mice by using the Cre recombinase fused to a mutated ligand binding domain of the human estrogen receptor (Cre-ERT), which binds tamoxifen but not estrogens. DNA excision was induced in a number of tissues after administration of tamoxifen to transgenic mice expressing Cre-ERT under the control of the cytomegalovirus promoter. However, the efficiency of excision varied between tissues, and the highest level ( approximately 40%) was obtained in the skin. To determine the efficiency of excision mediated by Cre-ERT in a given cell type, we have now crossed Cre-ERT-expressing mice with reporter mice in which expression of Escherichia coli beta-galactosidase can be induced through Cre-mediated recombination. The efficiency and kinetics of this recombination were analyzed at the cellular level in the epidermis of 6- to 8-week-old double transgenic mice. We show that site-specific excision occurred within a few days of tamoxifen treatment in essentially all epidermis cells expressing Cre-ERT. These results indicate that cell-specific expression of Cre-ERT in transgenic mice can be used for efficient tamoxifen-dependent, Cre-mediated recombination at loci containing loxP sites to generate site-specific somatic mutations in a spatio-temporally controlled manner.

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Figures

Figure 1

Figure 1

Epidermal stratification of mouse skin. Toluidine blue-stained, 2-μm, semi-thin section of newborn dorsal skin. A diagrammatic representation is given on the right side. Note that mitosis is restricted to the proliferative basal cells. (Bar = 25 μm.)

Figure 2

Figure 2

Pattern of Cre-ERT expression in the tail epidermis of double transgenic mice. Immunohistochemistry with anti-Cre antibody was performed on sections (10 μm-thick) of tail biopsies of 6- to 8-week-old wild-type (WT; a, _a_′) and CMV-Cre-ERT/ACZL double heterozygous transgenic mice (b_–_f, _b_′–_f_′). Double transgenic mice were injected for 5 consecutive days (days 0–4) with tamoxifen (1 mg/day). Sections were stained with DAPI and anti-Cre antibody. DAY 0 (b, _b_′), before the first tamoxifen injection; DAY 1 (c, _c_′) and DAY 3 (d, _d_′), after 1 and 3 days of tamoxifen treatment, respectively; DAY 7 (e, _e_′) and DAY 10 (f, _f_′), 3 and 6 days after the last tamoxifen injection, respectively. The cyan color corresponds to the DAPI staining (a_–_f), and the red color corresponds to the staining of Cre-ERT (a_–_f, _a_′–_f_′). B, S, and G, basal, spinous, and granular layers, respectively (see Fig. 1). (Bar = 25 μm.)

Figure 3

Figure 3

Kinetics of β-galactosidase expression in tail epidermis granular layer of CMV-Cre-ERT/ACZL double heterozygous mice. CMV-Cre-ERT/ACZL double heterozygous mice (6–8 weeks old) were injected daily with tamoxifen from day 0 to 4 (see legend to Fig. 2). Tail biopsies were collected just before the first tamoxifen injection (DAY 0, a) and at DAYS 1–5 (b_–_f) and treated as described in Materials and Methods (2-μm, semi-thin sections). Arrows point to the basement membrane (BM). B, S, G, and C, basal, spinous, granular, and cornified layers, respectively (see Fig. 1). (Bar = 25 μm.)

Figure 4

Figure 4

Vertical migration of granular layer-restricted β-galactosidase expression in tail epidermis of CMV-Cre-ERT/ACZL double heterozygous mice. Two series of daily tamoxifen injection, from day 0 to 4 and from day 25 to 29, were administered to 6- to 8-week-old CMV-Cre-ERT/ACZL double heterozygous mice. Tail biopsies were collected just before the first tamoxifen injection (DAY 0, a) and at different days after the first injection, as indicated (b_–_f). BM arrows, B, S, G, and C are as defined in legend to Fig. 3. (Bar = 25 μm.)

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