103D1.RNA Interference in C. elegansLab Overview:You will perform RNA interference (RNAi) in Caenorhabditis elegans(a nematode worm) using an RNAi “feeding” technique. In this technique, DE3 E .coli, that can make T7 RNA polymerase, will be used to produce. First you will need to spread E. colibacteria that have been transformed with the RNAi plasmids onto Nematode Agar plates. One of the RNAi plasmids produces double-stranded Unc-22RNA upon induction with IPTG. Another of the RNAi plasmids produces double-stranded lsy-2 RNA upon induction with IPTG. Also one of the plasmids is the empty RNAi vector. This week you will transfer C. elegans onto the plates where they will consume the E.coliand uptake the dsRNA. You also need to mark your chemotaxis plates for next week’s analysis. BackgroundThe soil nematode, Caenorhabditiselegans(C. elegans), is a well-studied laboratory model organism that offers a great potential for genetic analysis, partly because of its rapid (3-day) life cycle, small size (1.5-mm-long adult), and ease of laboratory cultivation. Its name is derived from latin: Caenomeaning “recent”, rhabditismeaning “rod-like”, and elegansmeaning “nice or beautiful”. Thousands of these animals can be grown on a single Petri dish seeded with a lawn of Escherichia colias the food source. C. eleganscomes in two sexes: a male and a hermaphrodite. The males represent less than one-tenth of a percent in a typical population of C. elegans, and are difficult to find on a dish. The hermaphrodite can reproduce by self-fertilization. Therefore only a single hermaphrodite worm is needed to start or maintain a population. A single worm can produce 300 - 350 offspring in its short lifespan. The entire C. elegansgenome was sequenced in 1998, making the C. elegansthe first multi-cellular organism to have its genome fully sequenced. The genome sequence data have emphasized the considerable conservation of biological mechanisms and processes across the animal kingdom as a model system has contributed significantly to our biological understanding of many organisms including humans. By the 1990’s, researchers surprisingly discovered that both sense and anti-sense RNA transcripts and double stranded RNAs led to apparent gene inactivation in a number of organisms. The inhibitory effect of double-stranded RNA was studied in C. elegansand *Read "the Lab Overview” of Lab D1 and 2.State lab Goal(s). Outline or make a flow chart of the methods.
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