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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 homozygous mtK14neo pups. (A) Gross phenotype of a 1-d-old pup, showing severe blisters on the front legs, paws, and chest. (B) H&E staining of skin from a homozygous mtK14neo pup. The separation occurred in the basal cell layer within the epidermis. (C–E) Immunofluorescence microscopy of pup skin. The sections were stained for mutant K14 (Texas red, C–E), integrin α6 (FITC, D), and keratin 10 (FITC, E). Overlapping of K14 staining with either integrin α6 (D) or keratin 10 (E) results in yellow fluorescence. (F) AciI digestion of semi-quantitative RT-PCR products from mouse RNA. (1) Wild type; (2) homozygous mtK14neo; (3 and 4) +/mtK14neo; (5) +/mtK14loxP. Note the lower expression levels of mtK14neo allele in +/mtK14neo mice shown in lanes 3 and 4.
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Figure 2: Phenotype of homozygous mtK14neo pups. (A) Gross phenotype of a 1-d-old pup, showing severe blisters on the front legs, paws, and chest. (B) H&E staining of skin from a homozygous mtK14neo pup. The separation occurred in the basal cell layer within the epidermis. (C–E) Immunofluorescence microscopy of pup skin. The sections were stained for mutant K14 (Texas red, C–E), integrin α6 (FITC, D), and keratin 10 (FITC, E). Overlapping of K14 staining with either integrin α6 (D) or keratin 10 (E) results in yellow fluorescence. (F) AciI digestion of semi-quantitative RT-PCR products from mouse RNA. (1) Wild type; (2) homozygous mtK14neo; (3 and 4) +/mtK14neo; (5) +/mtK14loxP. Note the lower expression levels of mtK14neo allele in +/mtK14neo mice shown in lanes 3 and 4.

Mentions: Correctly targeted ES cells bearing the point mutation were injected into mouse blastocysts, and heterozygous mtK14neo (+/mtK14neo) mice were derived. These mice appeared normal with no gross skin phenotypes, and histological analysis did not show microscopic blisters (data not shown). This was unexpected as the equivalent mutation in humans results in a dominantly inherited disease. We hypothesized that the lack of a phenotype in these mice was due to an interference of the neo cassette with the expression of the mutant K14 gene. This hypothesis was supported by semi-quantitative RT-PCR analysis that showed a twofold lower expression of mtK14neo than wild-type K14 (Fig. 2 F).


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 homozygous mtK14neo pups. (A) Gross phenotype of a 1-d-old pup, showing severe blisters on the front legs, paws, and chest. (B) H&E staining of skin from a homozygous mtK14neo pup. The separation occurred in the basal cell layer within the epidermis. (C–E) Immunofluorescence microscopy of pup skin. The sections were stained for mutant K14 (Texas red, C–E), integrin α6 (FITC, D), and keratin 10 (FITC, E). Overlapping of K14 staining with either integrin α6 (D) or keratin 10 (E) results in yellow fluorescence. (F) AciI digestion of semi-quantitative RT-PCR products from mouse RNA. (1) Wild type; (2) homozygous mtK14neo; (3 and 4) +/mtK14neo; (5) +/mtK14loxP. Note the lower expression levels of mtK14neo allele in +/mtK14neo mice shown in lanes 3 and 4.
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Related In: Results  -  Collection

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Figure 2: Phenotype of homozygous mtK14neo pups. (A) Gross phenotype of a 1-d-old pup, showing severe blisters on the front legs, paws, and chest. (B) H&E staining of skin from a homozygous mtK14neo pup. The separation occurred in the basal cell layer within the epidermis. (C–E) Immunofluorescence microscopy of pup skin. The sections were stained for mutant K14 (Texas red, C–E), integrin α6 (FITC, D), and keratin 10 (FITC, E). Overlapping of K14 staining with either integrin α6 (D) or keratin 10 (E) results in yellow fluorescence. (F) AciI digestion of semi-quantitative RT-PCR products from mouse RNA. (1) Wild type; (2) homozygous mtK14neo; (3 and 4) +/mtK14neo; (5) +/mtK14loxP. Note the lower expression levels of mtK14neo allele in +/mtK14neo mice shown in lanes 3 and 4.
Mentions: Correctly targeted ES cells bearing the point mutation were injected into mouse blastocysts, and heterozygous mtK14neo (+/mtK14neo) mice were derived. These mice appeared normal with no gross skin phenotypes, and histological analysis did not show microscopic blisters (data not shown). This was unexpected as the equivalent mutation in humans results in a dominantly inherited disease. We hypothesized that the lack of a phenotype in these mice was due to an interference of the neo cassette with the expression of the mutant K14 gene. This hypothesis was supported by semi-quantitative RT-PCR analysis that showed a twofold lower expression of mtK14neo than wild-type K14 (Fig. 2 F).

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