Epidermolysis Bullosa Simplex (EBS) is a rare human genetic skin disorder that results from mutations in the keratin 14, keratin 5, or plectin genes, and occurs in 1 out of 20 000 live births. In most cases, EBS is caused by dominant-negative missense mutations in the cytokeratin 5 (K5) or 14 (K14) genes. The primary symptom of EBS is the formation of blisters on the hands and feet of affected individuals. This project will focus on creating a zebrafish model for the disease using a two point mutation of keratin 5, identified to be linked with EBS in humans through the I161S and E477K mutations. The I161S and E477K mutants have been linked with mild and severe forms of the disease, respectively. Both of these mutations are found in the central rod domain of the keratin, which is involved in keratin assembly. The zebrafish and human keratin 5 proteins share an 83.2 percent similarity in this domain. Moreover, the isoleucine (I161) and glutamic acid (E477) are also conserved. Keratin 5 is ubiquitously expressed in the surface epithelial tissue of zebrafish embryos, as in humans. Using site-directed mutagenesis, we inserted point mutations in the zebrafish krt5 cDNA to mimic the I161S and E477K mutations observed in humans. Our lab has recently characterized a cis-acting regulatory element specific to the ectoderm of the median and pectoral fin fold of developing zebrafish. To ensure embryo survival, we have used these elements to ectopically express mutant keratin 5 only in the fin fold ectoderm. Mutant keratin 5 is linked to an eGFP reporter gene via the 2A peptide for screening purposes. Multiple transgenic lines for the I161S mutation, Tg(epi+β-globin:ker5 I161S-2A-eGFP), have recently been established. Preliminary phenotypic analysis shows little to no blistering. However, trauma or friction may cause blisters to form, as is the case in many forms of EB in humans. In an attempt to induce blistering, the zebrafish are currently being observed under different environmental stressors. Simple microscopy will be used to detect phenotypic defects in the skin, such as the formation of blisters. Further phenotypical analysis will include the structural integrity of the fin and the potential formation of keratin aggregates. The integrity of the keratin filament network in affected fins compared to wild type fins will be determined using atomic force microscopy. We will examine the keratin network and the possibility of keratin aggregates using immunohistochemistry and electron microscopy on the mutant fish as well as wild type fish. Our goal is to create a disease model for EBS that will be used for high throughput drug screening to identify candidate drugs to cure or alleviate the symptoms of EBS.