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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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Targeting strategy and Southern blot analysis. (A) Targeting strategy. Open boxes represent the exons. The asterisk denotes the C→T point mutation, and neo denotes the neomycin-resistance selection cassette. (B) Southern blot analysis of EcoRV-digested genomic DNA. The 5′ external probe (A) hybridized to an 11.7-kb fragment from the wild-type K14 locus, and a 5.5-kb fragment from the mtK14neo locus. The 3′ external probe (A) hybridized to the same 11.7-kb fragment from the wild-type K14 locus, and an 8.0-kb fragment from the mtK14neo locus. het., heterozygote; homo., homozygote.
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Figure 1: Targeting strategy and Southern blot analysis. (A) Targeting strategy. Open boxes represent the exons. The asterisk denotes the C→T point mutation, and neo denotes the neomycin-resistance selection cassette. (B) Southern blot analysis of EcoRV-digested genomic DNA. The 5′ external probe (A) hybridized to an 11.7-kb fragment from the wild-type K14 locus, and a 5.5-kb fragment from the mtK14neo locus. The 3′ external probe (A) hybridized to the same 11.7-kb fragment from the wild-type K14 locus, and an 8.0-kb fragment from the mtK14neo locus. het., heterozygote; homo., homozygote.

Mentions: To generate a mouse model that mimics EBS-DM at the genetic level, we introduced a point mutation in codon 131 (equivalent to arginine 125 in human K14) of the mouse K14 gene. The gene targeting vector used to introduce this mutation into ES cells also contained a neo cassette in intron I (Fig. 1 A). The linearized targeting vector was electroporated into ES cells, and homologous recombination was identified by Southern blot analysis of genomic DNA from these cells (Fig. 1 B). The presence of the point mutation was confirmed by sequence analysis (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)

Targeting strategy and Southern blot analysis. (A) Targeting strategy. Open boxes represent the exons. The asterisk denotes the C→T point mutation, and neo denotes the neomycin-resistance selection cassette. (B) Southern blot analysis of EcoRV-digested genomic DNA. The 5′ external probe (A) hybridized to an 11.7-kb fragment from the wild-type K14 locus, and a 5.5-kb fragment from the mtK14neo locus. The 3′ external probe (A) hybridized to the same 11.7-kb fragment from the wild-type K14 locus, and an 8.0-kb fragment from the mtK14neo locus. het., heterozygote; homo., homozygote.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195993&req=5

Figure 1: Targeting strategy and Southern blot analysis. (A) Targeting strategy. Open boxes represent the exons. The asterisk denotes the C→T point mutation, and neo denotes the neomycin-resistance selection cassette. (B) Southern blot analysis of EcoRV-digested genomic DNA. The 5′ external probe (A) hybridized to an 11.7-kb fragment from the wild-type K14 locus, and a 5.5-kb fragment from the mtK14neo locus. The 3′ external probe (A) hybridized to the same 11.7-kb fragment from the wild-type K14 locus, and an 8.0-kb fragment from the mtK14neo locus. het., heterozygote; homo., homozygote.
Mentions: To generate a mouse model that mimics EBS-DM at the genetic level, we introduced a point mutation in codon 131 (equivalent to arginine 125 in human K14) of the mouse K14 gene. The gene targeting vector used to introduce this mutation into ES cells also contained a neo cassette in intron I (Fig. 1 A). The linearized targeting vector was electroporated into ES cells, and homologous recombination was identified by Southern blot analysis of genomic DNA from these cells (Fig. 1 B). The presence of the point mutation was confirmed by sequence analysis (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