Gen875 RNAi discussion - Gen 875 Discussion Functional...

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Gen 875 Discussion Functional Genomics using RNAi Presented by Young-Jun Choi & Hui Cai
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Genetics Screening A genetic screen is a procedure to identify and select individuals who possess a phenotype of interest. Forward genetic screens Find the genetic basis of a phenotype Mutations are generated at random, phenotypes of interest are scored, and the mutated gene is identified. Limited by mutagenesis techniques, the large numbers of mutants that must be analysed, multiple functions that a gene may have. Reverse genetic screens Find the possible phenotypes that may derive from a specific genetic sequence Random or directed deletions, insertions and point mutations Gene silencing: RNA interference
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RNA interference (RNAi) Based on the ability of double-stranded RNAs (dsRNA), small interfering RNAs (siRNAs), or small hairpin RNAs (shRNAs) to silence a target gene through the specific destruction of that gene’s mRNA. RNAi pathway is conserved in most eukaryotic organisms Dr Andrew Fire at Stanford University and Dr. Craig Mello at the University of Massachusetts published their study on the mechanism of RNAi in Nature in 1998 They received Nobel Prize in Physiology or Medicine in 2006
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Discovery of RNAi by Fire and Mello Tested phenotypic effect of RNA injected into C. elegans Annealed sense/antisense RNA, but neither antisense nor sense RNA alone, caused the predicted phenotype Only injection of dsRNA led to an efficient loss of the target mRNA It appears that fly embryos were transformed with RNAi constructs. Is this the only way to introduce RNAi constructs into this organisms and, if so, how does this limit the use of RNAi in other organisms not so easily manipulated?
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RNA interference process and the biochemical machinery involved ds RNA is cut into short double-stranded fragments called small interfering RNAs ( siRNAs ) by Dicer , a ribonuclease III-like nuclease . siRNAs are then separated into single strands and integrated into an active RNA-induced silencing complex ( RISC ) The antisense strand is loaded into RISC and links the complex to the mRNA strand by base-pairing. The RISC complex cuts the mRNA strand, and the mRNA is subsequently degraded. • Exogenous, e.g. a virus with an RNA genome Endogenous, e.g. Stem-loop structure of pre-microRNAs
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Roles of RNAi pathway in the cell Protects against RNA virus infections, especially in plants and invertebrate animals Maintains genome stability by keeping mobile elements silent Represses protein synthesis and regulate the development of organisms MicroRNA (miRNA), a class of endogenous RNA -- regulate gene
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