Gene Delivery of Lamb3 To Cultured Keratinocyte Cells and Produce Packaging Cell Line

Background and aim: epidermolysis bullosa (eb) is a group of genetically inherited disease with clinically and genetically heterogeneousity, the main feature in all the paitents is development of painful blisters on the skin after any minor trauma. diagnosis of eb is confirmed by mutational analysis, transmission electron microscopy (tem), and immunofluorescence antigen mapping (ifm). the current international classification contains; eb simplex (ebs), junctional eb (jeb), dystrophic eb (deb), and kindler syndrome (ks). in jeb patients’ mutations in lamb3, collagen17 and integrin α6β4 are detected. all of these proteins have essential roles in hemidesmosomal junction, a cell-ecm junction, which is important for tissue structure and integrity, especially in stratified epithelia, like as a skin. laminin332 has a tetrameric structure that consists of lamb3, lama3 and lamc2. this protein plays an important role in the basement membrane of the epidermis. indeed, one of the most important components of fibers in basement membrane is lamb3 and helps hold the skin together. studies showed the importance of this protein in several other functions such as wound healing. the mutation in lamb3 detected in about 70% of jeb patients. the treatment of eb is primarily preventive and supportive but studying for better ways to treat and relieve the symptoms of epidermolysis bullosa continue. a novel approach for this disease is gene therapy. in this project we produce packaging cell line by gama-retrovirus and deliver the correct lamb3 gene to keratinocyte and restore this protein function that can use as a therapeutics method. flowcytometry and immunocytochemistry are tests for checking the expression of lamb3 gene.methods: we cultured the keratinocyte and use packaging cells, plat e and pg13 and transduced lamb3 viral vector to this cells. we check this test by flowcytometry and icc with lamb3 antibody.then we will transduced keratinocytes by this packaging cellsresults: the lamb3 gene expressed in pg13, and fibroblast cells detected by flowcytometry and immunocytochemistry testconclusion: we can use this packaging cell as a tool for gene delivery to human cells.