The Effect of Srb-16 and F35B1.3 RNAi Gene Treatment on Chemosensation in Caenorhabditis elegans through Observation of Chemotaxis. Abstract:
The study of sensory systems in humans is a vital area of study to further pursue our knowledge of the mechanisms occurring in the nervous systems. However due to the complications surrounding human testing, model organisms such as C. elegans worms can be utilized as a comparison to create paradigms relevant to human sensory systems as the genetic basis for both organisms is related amongst other factors such as the worm’s rapid reproductive cycle. Using RNAi treatment on C. elegans we tested the effects of inhibiting the synthesis of particular proteins in relation to the organism’s ability to detect and consequently move towards – that is chemosensation and chemotaxis - characteristically favorable odorants. This type of work hopes to build a basis for understanding the network of chemosensory signaling systems in humans and the corresponding proteins produced by particular gene sequences. Results suggested that gene sequence Srb-16 was not involved in encoding for a protein involved in chemosensation although contrary to previous experiments. The second gene sequence that was analyzed, F35B1.3- a mostly unknown sequence- was shown to likely encode for a protein in the sensory olfactory system as characteristic chemosensation behavior of C. elegans was altered. Introduction:
This experiment was carried out to test the role of gene sequences Srb-16 and F34B1.3 in the chemosensation of C. elegans. Using RNAi treatment, the genes were inhibited from encoding their specific proteins and the resulting effect was analyzed to determine whether or not the genes play a role in chemotaxis of diacetyl. Diacetyl (DA) is a toxic buttery smelling odorant normally attractive to C. elegans. By observing the worms post RNAi treatment in their ability to detect and move towards DA, conclusions were drawn as to whether the gene sequence encodes for proteins that are part of the organism’s olfactory chemosensation systems or proteins involved in the subsequent motor processes that are in parallel with detection of an odorant. Method:
We prepared four agarose plates – two controls and two experimental – each with a bacterial lawn containing four different RNAi clones. A negative control containing RNAi gene sequence L4440; a positive control, with gene ODR-10; an experimental with gene Srb-16 and a second experimental with gene F53B1.3. Worms were obtained from the teaching assistant that had already been prepared so that all worms are present at the same stage of the C. elegans lifecycle. Approximately two worms were then added to each plate to be RNAi treated with different gene sequences. The worms were taken from a diluted solution of multiple worms so that a 2 micro liter measurement yielded approximately two worms if mixed and pipetted from appropriately. Each RNAi agarose treatment plate had optimally two worms placed at the inner edge of the bacterial lawn. To ensure that at least one and no more than four worms were added to each plate we were instructed to observe the plate under a dissecting microscope and record how many had been added. Each plate was labeled on the agar side –before adding the worms- with their specific RNAi treatment, student name, date, etc. Finally the plates were turned into the lab technician for incubation at 15 degrees Celsius for 7 days to develop a successive generation that we would test the following week for affects on chemotaxis. Week two involved the testing of our RNAi treated worms on agar chemotaxis plates. Agar plates were prepared by drawing a line beneath the plate splitting it into two semi circles. A circle in the center of the plate about the size of a filter-screen was also added along with two smaller circles equal distance from the center. One of these circles was labeled DA (for diacetyl odorant) and the other labeled 0 (for no odorant...
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