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An inducible mouse model for epidermolysis bullosa simplex: implications for gene therapy.

Cao T, Longley MA, Wang XJ, Roop DR - J. Cell Biol. (2001)

Bottom Line: This observation provides an explanation for the lack of mosaic forms of EBS-DM.In addition, we show that decreased mutant keratin 14 expression resulted in normal morphology and functions of the skin.Our results have important implications for gene therapy of EBS and other dominantly inherited diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.

ABSTRACT
The Dowling-Meara variant of epidermolysis bullosa simplex (EBS-DM) is a severe blistering disease inherited in an autosomal-dominant fashion. Here we report the generation of a mouse model that allows focal activation of a mutant keratin 14 allele in epidermal stem cells upon topical administration of an inducer, resulting in EBS phenotypes in treated areas. Using laser capture microdissection, we show that induced blisters healed by migration of surrounding nonphenotypic stem cells into the wound bed. This observation provides an explanation for the lack of mosaic forms of EBS-DM. In addition, we show that decreased mutant keratin 14 expression resulted in normal morphology and functions of the skin. Our results have important implications for gene therapy of EBS and other dominantly inherited diseases.

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Induction and characterization of RU486 induced blisters. (A) Gross phenotype of an induced blister on a mtK14neo/CrePR1 pup after two RU486 treatments on the right front leg. No blisters developed on the untreated leg (A) or in +/mtK14neo or CrePR1 control pups treated with RU486 (data not shown). (B) The right front paw of a mtK14neo/CrePR1 pup 10 d after blister formation upon treatment. (C) The left front paw and leg of a mtK14neo/CrePR1 mouse 6 mo after blister formation and cessation of RU486 treatment. The blistered area, including the palm and leg, healed without scarring, and there is normal hair growth on the leg. No additional blisters formed without further RU486 treatment. (D and E) H&E staining (D) and immunofluorescence microscopy with an anti-K14 antibody (Texas red, E) of an induced blister edge. Blistering occurred in the basal keratinocytes (arrowheads) on the chest (D and E), front leg, and paw (data not shown). The asterisk denotes cytolysis, and dashed lines outline the hair follicles. (F and G) PCR analysis of DNA for the presence of the loxP site (F) and neo cassette (G). (1–3) Epidermal DNA samples from different areas of a treated mtK14neo/CrePR1 pup. (1) Untreated area; (2) blister roof; (3) healed blistered area 1 wk after blister formation. (4–6) DNA samples from control mice. (4) Wild type; (5) +/mtK14loxP; (6) +/mtK14neo.
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Figure 4: Induction and characterization of RU486 induced blisters. (A) Gross phenotype of an induced blister on a mtK14neo/CrePR1 pup after two RU486 treatments on the right front leg. No blisters developed on the untreated leg (A) or in +/mtK14neo or CrePR1 control pups treated with RU486 (data not shown). (B) The right front paw of a mtK14neo/CrePR1 pup 10 d after blister formation upon treatment. (C) The left front paw and leg of a mtK14neo/CrePR1 mouse 6 mo after blister formation and cessation of RU486 treatment. The blistered area, including the palm and leg, healed without scarring, and there is normal hair growth on the leg. No additional blisters formed without further RU486 treatment. (D and E) H&E staining (D) and immunofluorescence microscopy with an anti-K14 antibody (Texas red, E) of an induced blister edge. Blistering occurred in the basal keratinocytes (arrowheads) on the chest (D and E), front leg, and paw (data not shown). The asterisk denotes cytolysis, and dashed lines outline the hair follicles. (F and G) PCR analysis of DNA for the presence of the loxP site (F) and neo cassette (G). (1–3) Epidermal DNA samples from different areas of a treated mtK14neo/CrePR1 pup. (1) Untreated area; (2) blister roof; (3) healed blistered area 1 wk after blister formation. (4–6) DNA samples from control mice. (4) Wild type; (5) +/mtK14loxP; (6) +/mtK14neo.

Mentions: Mice carrying the CrePR1 transgene under the control of a K5 or K14 promoter were bred with +/mtK14neo mice, and bigenic pups (mtK14neo/CrePR1) were obtained. RU486 (0.5 mg/ml in ethanol) was applied once daily to the forelimbs and chest of newborn pups, areas prone to mechanical trauma. After several (two to seven) days of RU486 treatment, we observed blisters filled with fluid on the front legs and paws of bigenic pups (Fig. 4 A). RU486 treatment was terminated once blisters became visible. Histological analysis and immunofluorescence microscopy revealed that blistering occurred within the basal layer of the epidermis, as expected (Fig. 4D and Fig. E).


An inducible mouse model for epidermolysis bullosa simplex: implications for gene therapy.

Cao T, Longley MA, Wang XJ, Roop DR - J. Cell Biol. (2001)

Induction and characterization of RU486 induced blisters. (A) Gross phenotype of an induced blister on a mtK14neo/CrePR1 pup after two RU486 treatments on the right front leg. No blisters developed on the untreated leg (A) or in +/mtK14neo or CrePR1 control pups treated with RU486 (data not shown). (B) The right front paw of a mtK14neo/CrePR1 pup 10 d after blister formation upon treatment. (C) The left front paw and leg of a mtK14neo/CrePR1 mouse 6 mo after blister formation and cessation of RU486 treatment. The blistered area, including the palm and leg, healed without scarring, and there is normal hair growth on the leg. No additional blisters formed without further RU486 treatment. (D and E) H&E staining (D) and immunofluorescence microscopy with an anti-K14 antibody (Texas red, E) of an induced blister edge. Blistering occurred in the basal keratinocytes (arrowheads) on the chest (D and E), front leg, and paw (data not shown). The asterisk denotes cytolysis, and dashed lines outline the hair follicles. (F and G) PCR analysis of DNA for the presence of the loxP site (F) and neo cassette (G). (1–3) Epidermal DNA samples from different areas of a treated mtK14neo/CrePR1 pup. (1) Untreated area; (2) blister roof; (3) healed blistered area 1 wk after blister formation. (4–6) DNA samples from control mice. (4) Wild type; (5) +/mtK14loxP; (6) +/mtK14neo.
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Related In: Results  -  Collection

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Figure 4: Induction and characterization of RU486 induced blisters. (A) Gross phenotype of an induced blister on a mtK14neo/CrePR1 pup after two RU486 treatments on the right front leg. No blisters developed on the untreated leg (A) or in +/mtK14neo or CrePR1 control pups treated with RU486 (data not shown). (B) The right front paw of a mtK14neo/CrePR1 pup 10 d after blister formation upon treatment. (C) The left front paw and leg of a mtK14neo/CrePR1 mouse 6 mo after blister formation and cessation of RU486 treatment. The blistered area, including the palm and leg, healed without scarring, and there is normal hair growth on the leg. No additional blisters formed without further RU486 treatment. (D and E) H&E staining (D) and immunofluorescence microscopy with an anti-K14 antibody (Texas red, E) of an induced blister edge. Blistering occurred in the basal keratinocytes (arrowheads) on the chest (D and E), front leg, and paw (data not shown). The asterisk denotes cytolysis, and dashed lines outline the hair follicles. (F and G) PCR analysis of DNA for the presence of the loxP site (F) and neo cassette (G). (1–3) Epidermal DNA samples from different areas of a treated mtK14neo/CrePR1 pup. (1) Untreated area; (2) blister roof; (3) healed blistered area 1 wk after blister formation. (4–6) DNA samples from control mice. (4) Wild type; (5) +/mtK14loxP; (6) +/mtK14neo.
Mentions: Mice carrying the CrePR1 transgene under the control of a K5 or K14 promoter were bred with +/mtK14neo mice, and bigenic pups (mtK14neo/CrePR1) were obtained. RU486 (0.5 mg/ml in ethanol) was applied once daily to the forelimbs and chest of newborn pups, areas prone to mechanical trauma. After several (two to seven) days of RU486 treatment, we observed blisters filled with fluid on the front legs and paws of bigenic pups (Fig. 4 A). RU486 treatment was terminated once blisters became visible. Histological analysis and immunofluorescence microscopy revealed that blistering occurred within the basal layer of the epidermis, as expected (Fig. 4D and Fig. E).

Bottom Line: This observation provides an explanation for the lack of mosaic forms of EBS-DM.In addition, we show that decreased mutant keratin 14 expression resulted in normal morphology and functions of the skin.Our results have important implications for gene therapy of EBS and other dominantly inherited diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.

ABSTRACT
The Dowling-Meara variant of epidermolysis bullosa simplex (EBS-DM) is a severe blistering disease inherited in an autosomal-dominant fashion. Here we report the generation of a mouse model that allows focal activation of a mutant keratin 14 allele in epidermal stem cells upon topical administration of an inducer, resulting in EBS phenotypes in treated areas. Using laser capture microdissection, we show that induced blisters healed by migration of surrounding nonphenotypic stem cells into the wound bed. This observation provides an explanation for the lack of mosaic forms of EBS-DM. In addition, we show that decreased mutant keratin 14 expression resulted in normal morphology and functions of the skin. Our results have important implications for gene therapy of EBS and other dominantly inherited diseases.

Show MeSH
Related in: MedlinePlus