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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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Phenotype of +/mtK14loxP pups. (A) Gross phenotype of a 2-d-old pup, showing blisters on the left front leg and abdomen. (B–G) Transmission electron microscopy of skin samples from +/mtK14loxP pups (B, C, and E–G) and normal littermate (D). The mtK14loxP pup skin showed keratin clumps and short keratin filaments in the basal keratinocytes (B, E–G), but normal keratin filaments in the suprabasal layer of the epidermis (B and C). Normal skin showed long keratin filaments in the basal keratinocytes (D). Kc, keratin clumps; Kf, keratin filaments; Kg, keratohyalin granules; Nu, nucleus. Small arrowheads denote hemidesmosomes, indicating the position of the basement membrane. Large arrowheads denote desmosomes. Bars: (B) 5 μm; (C) 100 nm; (D–G) 1 μm.
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Figure 3: Phenotype of +/mtK14loxP pups. (A) Gross phenotype of a 2-d-old pup, showing blisters on the left front leg and abdomen. (B–G) Transmission electron microscopy of skin samples from +/mtK14loxP pups (B, C, and E–G) and normal littermate (D). The mtK14loxP pup skin showed keratin clumps and short keratin filaments in the basal keratinocytes (B, E–G), but normal keratin filaments in the suprabasal layer of the epidermis (B and C). Normal skin showed long keratin filaments in the basal keratinocytes (D). Kc, keratin clumps; Kf, keratin filaments; Kg, keratohyalin granules; Nu, nucleus. Small arrowheads denote hemidesmosomes, indicating the position of the basement membrane. Large arrowheads denote desmosomes. Bars: (B) 5 μm; (C) 100 nm; (D–G) 1 μm.

Mentions: To unequivocally demonstrate that the low expression level of the mutant K14 allele led to the absence of a phenotype in +/mtK14neo mice, we deleted the neo cassette in mtK14neo ES cell clones via Cre-mediated excision (Fig. 1 A). Heterozygous mtK14loxP (+/mtK14loxP) pups derived from these ES cells spontaneously developed large blisters on their forelimbs and trunk (Fig. 3 A), as expected for this dominant mutation. Semi-quantitative RT-PCR analysis of RNA isolated from these pups showed comparable expression levels of the mutant and wild-type K14 alleles (Fig. 2 F). Histological analysis and immunofluorescence microscopy revealed similar findings as in the homozygous mtK14neo pups (data not shown).


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

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

Phenotype of +/mtK14loxP pups. (A) Gross phenotype of a 2-d-old pup, showing blisters on the left front leg and abdomen. (B–G) Transmission electron microscopy of skin samples from +/mtK14loxP pups (B, C, and E–G) and normal littermate (D). The mtK14loxP pup skin showed keratin clumps and short keratin filaments in the basal keratinocytes (B, E–G), but normal keratin filaments in the suprabasal layer of the epidermis (B and C). Normal skin showed long keratin filaments in the basal keratinocytes (D). Kc, keratin clumps; Kf, keratin filaments; Kg, keratohyalin granules; Nu, nucleus. Small arrowheads denote hemidesmosomes, indicating the position of the basement membrane. Large arrowheads denote desmosomes. Bars: (B) 5 μm; (C) 100 nm; (D–G) 1 μm.
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Related In: Results  -  Collection

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Figure 3: Phenotype of +/mtK14loxP pups. (A) Gross phenotype of a 2-d-old pup, showing blisters on the left front leg and abdomen. (B–G) Transmission electron microscopy of skin samples from +/mtK14loxP pups (B, C, and E–G) and normal littermate (D). The mtK14loxP pup skin showed keratin clumps and short keratin filaments in the basal keratinocytes (B, E–G), but normal keratin filaments in the suprabasal layer of the epidermis (B and C). Normal skin showed long keratin filaments in the basal keratinocytes (D). Kc, keratin clumps; Kf, keratin filaments; Kg, keratohyalin granules; Nu, nucleus. Small arrowheads denote hemidesmosomes, indicating the position of the basement membrane. Large arrowheads denote desmosomes. Bars: (B) 5 μm; (C) 100 nm; (D–G) 1 μm.
Mentions: To unequivocally demonstrate that the low expression level of the mutant K14 allele led to the absence of a phenotype in +/mtK14neo mice, we deleted the neo cassette in mtK14neo ES cell clones via Cre-mediated excision (Fig. 1 A). Heterozygous mtK14loxP (+/mtK14loxP) pups derived from these ES cells spontaneously developed large blisters on their forelimbs and trunk (Fig. 3 A), as expected for this dominant mutation. Semi-quantitative RT-PCR analysis of RNA isolated from these pups showed comparable expression levels of the mutant and wild-type K14 alleles (Fig. 2 F). Histological analysis and immunofluorescence microscopy revealed similar findings as in the homozygous mtK14neo pups (data not shown).

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