The ability to specifically express or delete single gene products within cells is a means of determining gene function. In contrast to cell lines, however, introduction of DNA or RNA into normal cultured keratinocytes is difficult. Viral delivery methods are currently the most effective means of long-term gene overexpression or silencing in keratinocytes in vitro. Recently, new methods have been developed to generate viruses for delivery of various types of RNA sequences such as small hairpin (sh-RNA) and microRNA (miRNA). Since retroviruses and lentiviruses have the ability to integrate into the cell genome, they have become the preferable tool for delivery of exogenous cDNAs and RNAs into keratinocytes. We have adopted, modified and developed a wide range of techniques to use lenti- and retro-viruses to infect different types of epithelial cells in vitro, including primary and transformed keratinocytes. We generated viral expression cassettes that incorporate polymerase type II or polymerase type III promoters necessary for expression of gene cDNAs and RNA sequences. The DNA/RNA Delivery Core will support NU SDRC researchers and attract new investigators in the area of keratinocyte biology by providing service and/or instruction about basic techniques for the development and use of siRNA/shRNA/miRNA and cDNA in experiments involving keratinocytes. This core will also provide NU SDRC researchers with assistance in the use of viral methods for infecting keratinocytes, primarily focusing on retro- and lentiviruses. The Core will offer the expertise and instruction needed for the successful gene overexpression/silencing proposed in the Pilot and Feasibility projects. Assistance in the selection of target sequences and design of primers as well as viral constructs were indicated to be of greatest general interest for the SDRC investigators, and thus is the major service of this Core. Helping SDRC members troubleshoot the steps in the generation of stably infected cell cultures is an important role for the Core. Dissemination of the information about changes in technology through the SDRC Core-directed Website and Newsletter, and the Enrichment Program's Workshop series will enable SDRC investigators to perform their experiments at the most advanced """"""""state-of-the-art"""""""" level.
Introduction of DNA and RNA into keratinocytes has been a useful biologic tool for discovering gene function. Keratinocytes are notoriously difficult to transfect and thus require infection as a means of delivery. The DNA/RNA Delivery Core will train, consult, and assist epithelial biologists of the SDRC in these techniques. This Core will greatly facilitate studies in keratinocyte biology.
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