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Gene Expression Profiling of Mouse Epidermal Keratinocytes

DNA microarray technology is a powerful tool for genome-wide gene expression analysis of biological samples including skin and epidermal keratinocytes. Here we review the methodology for expression profiling analysis of skin tissue or purified keratinocytes from mice. We expla ...

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Analysis of Tissue-Specific Gene Expression Using Laser Capture Microdissection

Epithelial tissues exhibit optimal conditions for studying cellular differentiation since the differentiation status of a single cell can be determined by its distance to the basal membrane. For that reason Laser Capture Microdissection (LCM) may serve as a perfect tool to compare the c ...

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Comprehensive Transcriptional Profiling of Human Epidermis, Reconstituted Epidermal Equivalents, and Cultured Keratinocytes Using DNA Microarray Chips

Because of its accessibility, skin has been among the first organs analyzed using DNA microarrays; psoriasis, melanomas, carcinomas, chronic wound biopsies, and epidermal keratinocytes in culture have been intensely investigated. Skin has everything: stem cells, differentia ...

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Embryonic Mammary Anlagen Analysis Using Immunolabelling of Whole Mounts

The mouse mammary gland is a unique organ since although the mammary gland primordium forms during embryogenesis, the majority of development occurs postnatally upon hormonal stimulation at puberty and full functionality (i.e. lactation) is not achieved until after parturition. S ...

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Detection of Gene Expression in Embryonic Tissues and Stratified Epidermis by In Situ Hybridization

Although recent molecular biology advances provide a very effective way in determining localized gene expression, the visualization of the expression by in situ hybridization of whole-mount embryos or paraffin-embedded tissue sections continues to be an excellent method to det ...

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Molecular Profiling of the Epidermis: A Proteomics Approach

Mouse skin has been used extensively as a model system to study the development of cancer. Recent emphasis has been focused on elucidating the molecular mechanisms by which chemical carcinogens or tumor promoters cause the growth of cutaneous malignancies. In this regard, many transgenic ...

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Whole-Mount Assays for Gene Induction and Barrier Formation in the Developing Epidermis

The skin as a surface organ is uniquely accessible for whole embryo/foetal analyses of developmental changes, such as gene induction, protein expression, formation of epidermal-derived appendages such as hair follicles and formation of the protective barrier. Such analyses have em ...

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Isolation of Adult Mouse Stem Keratinocytes Using Magnetic Cell Sorting (MACS)

The functional and molecular characterization of mouse epidermal stem cells (SC) requires in many circumstances arduous technologies and elaboration of complicated transgenic mouse models. To avoid these difficulties, we have developed a simplified protocol to obtain prima ...

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A Versatile Murine 3D Organotypic Model to Evaluate Aspects of Wound Healing and Epidermal Organization

Three-dimensional (3D) organotypic models are increasingly being used to study aspects of epidermal organisation and cutaneous wound-healing events. These are largely dependent on laborious histological analysis and immunohistochemical approaches. Here we outline a me ...

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Tetracycline-Regulated Gene Expression in Transgenic Mouse Epidermis

The ability to specifically manipulate gene expression in vivo using mouse models has been one of the most important advances in understanding gene function over the last few decades. Methods used to control gene activities in the mouse include gene targeting and transgenic approaches. Wh ...

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Optical and Biochemical Dissection of Connexin and Disease-Linked Connexin Mutants in 3D Organotypic Epidermis

The epidermis is a complex tissue composed principally of differentiated keratinocytes that form a keratinized stratified squamous epithelium. The gap junction proteins, connexins (Cx), are differentially expressed throughout the stratified layers of the epidermis and the ...

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Cytokine Release in Tissue-Engineered Epidermal Equivalents After Prolonged Mechanical Loading

Prolonged mechanical loading of soft tissues may result in degeneration of these tissues, resulting in formation of pressure ulcers. The risk assessment of individuals might be improved by including measurements of the tissue response to mechanical loading. Cytokines, which are rel ...

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In Vivo Transplantation of Genetically Modified Mouse Embryonic Epidermis

Situations in which epidermal mutant mice display early lethality after birth are rather frequent. This condition precludes any kind of analysis of adult or even newborn mice tissues. We propose the in vivo embryonic skin transplantation as an alternative to solve this problem. This method ...

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Three-Dimensional Human Tissue Models of Wounded Skin

Human skin equivalents (HSEs) are in vitro tissues in which a fully differentiated, stratified squamous epithelium is grown at an air–liquid interface on a Type I collagen gel harboring human dermal fibroblasts. HSEs now provide experimental human tissue models to study factors that dire ...

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Identification of Epithelial Stem Cells In Vivo and In Vitro Using Keratin 19 and BrdU

Progress in the identification of skin stem cells and the improvement of culture methods open the possibility to use stem cells in regenerative medicine. Based on their quiescent nature, the development of label retention assays allowed the localization of skin stem cells in the bulge region ...

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A Transplant Model for Human Epidermal Skin Regeneration

This protocol describes a technically simple in vivo assay of long-term skin regeneration in human skin, providing a reliable method for epidermal tissue reconstitution using small numbers of several types of epithelial cells, from epithelial cell lines to primary epithelial stem ce ...

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Isolation and Culture of Hair Follicle Pluripotent Stem (hfPS) Cells and Their Use for Nerve and Spinal Cord Regeneration

The hair follicle is dynamic, cycling between growth (anagen), regression (catagen), and resting (telogen) phases throughout life. We have demonstrated that nestin-expressing hair follicle stem cells give rise to follicle structures during early anagen or growth phase of the hair fol ...

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Functional Investigations of Keratinocyte Stem Cells and Progenitors at a Single-Cell Level Using Multiparallel Clonal Microcultures

The basal layer of human interfollicular epidermis is thought to contain a minor compartment of quiescent or slowly cycling epithelial stem cells. These primitive keratinocytes give rise to the progenitors, which are the proliferating keratinocytes and which can be defined as early to l ...

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Limiting Dilution Analysis of Murine Epidermal Stem Cells Using an In Vivo Regeneration Assay

Epidermal stem cells are of major importance for tissue homeostasis, wound repair, tumor initiation, and gene therapy. Here we describe an in vivo regeneration assay to test for the ability of keratinocyte progenitors to maintain an epidermis over the long-term in vivo. Limiting dilution an ...

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Growth and Stratification of Epithelial Cells in Minimal Culture Conditions

Biological risk management is required in modern tissue engineering. Particular attention should be paid to the culture medium and the scaffold used. In this perspective, it is important to define minimal culture conditions which allow proper growth and differentiation of epitheli ...

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