We a short lifespan, quick and plentiful reproduction cycles,

We can assess the loss of gene function due to lowered levels of network proteins by using the process of RNA interference (RNAi) to reduce, or knock down, specific proteins within the worms and test if chemosensory neurons and chemotaxis are still functioning. If a certain gene is knocked down the worms will not behave as expected and can be concluded that the specific gene plays a significant role in the worms olfactory sensing system. The genes being tested are ODR-10, L4440, MNP-1, and LPR-4, which allow us to determine whether transcription factors or proteins are required for normal response to odorants.Keywords: RNA interference, chemotaxis, chemosensory neurons, Caenorhabditis elegans, short interfering RNA, gene silencing, double stranded RNAIntroduction:The object of this study is to examine the role of certain proteins in the chemosensory, responses to olfactory molecules, and chemotaxis, referring to movement in response to chemical stimuli, systems of the worm like Caenorhabditis elegans (Macmillan 2017). One of the five majors senses, smell, is a key factor in the chemotaxic abilities of all living beings as the olfactory system allows organisms to react by migrating towards food sources and away from harmful substances including  heavy metals and acids (Grishok et al. 2000). They are also considered model organisms for this experiment, due to similarities to the human body, a short lifespan, quick and plentiful reproduction cycles, and a compact genome (Bargmann 2006). Caenorhabditis elegans’ short life span causes them to grow from a single-celled egg to an adult worm in a matter of three and a half days through four different stages (Macmillan 2017).Containing similar sequences to that of humans, C. elegans’ genomes are about thirty times smaller than humans’, but encode for nearly the same amount of proteins, resulting in about 35% similarity (Ahringer 2006). Compared to the genomes of other mammals, C.elegans contains more supposed olfactory receptor genes, suggesting that  the chemosensory system in nematodes may be possible due to a combination of complex smaller systems (Taniguchi et al. 2014). Our use of C.elegans in the experiment is vital due to their parallels to the human structure and function in genomes. C.elegans are genetically programmed to create proteins that either help to eat or avoid certain compounds by sensing the chemo-attraction cues AWA and AWC, which perceive attractant stimuli, and AWB, which perceive repellant stimuli (Macmillan 2017). The positive control has bacteria, E.Coli, that contains an RNAi molecule which interfere with the ODR-10 gene that we know helps worms sense the attractant, diacetyl leading to worms that will not be able to sense diacetyl due to reduced AWA responses (Taniguchi et al. 2014). RNAi (RNA interference) will be used to greatly reduce the expression of proteins and transmembrane receptors that are likely to be part of the system for sensing and responding to chemical signals (Macmillan 2017). C. elegans genes are knocked down when worm feed on the RNAi tagged E.Coli from the bacterial lawn, into each worm and the RNAi to most efficiently insert RNAi rather than injection or soaking (Grishok et al. 2000). RNAi uses molecules of RNA that interfere with a cell’s ability to translate mRNA into a protein (Hart 2006).. Co-suppression is also known to silence transposons therefore at least in a small part caused by the same molecular machinery, for example through RNA induced degradation of mRNA molecules (Ketting et al. 2000). RNAi technology can be used to identify and assess the probability that a certain gene, out of the thousands in the genome, potentially participate in disease phenotypes (RNAi Overview 1996).  Researchers are hoping to establish a connection between the exact cause of Alzheimer’s Disease by using RNAi technology to blocking expression of the specific gene and evaluate its response to chemical compounds or changes in signaling pathways that would hopefully become a cure (Sengupta et al. 1996). A network of receptors and G-proteins are required for chemosensing therefore the experimental genes MNP-1 and LPR-4 are believed to have no effect on the C. elegans’ ability to chemotaxate because lowering the levels of the network proteins will alter the ability of the worms to respond normally to chemical signals.Methods and Materials: This study was done in the University of California, Santa Barbara laboratory, BSIF 2211.  Use a dissecting microscope to identify the different stages, L1-L4, adult, or the worms life cycle (Macmillan 2017). Each RNAi plate contains agar topped by a bacterial lawn of E.Coli, tagged with gene specific RNAi constructs, to sustain and the worms. Pipette 5 ?l from the center of the 1.5 mL tube of L4 worms to the edge of the bacterial lawn of each RNAi plate and are counted to determine the number of worms transferred for incubation in each plate (Macmillan 2017). Our RNAi plates consist of the positive control: ODR-10, negative control: L4440, experimental gene #18 : MNP-1, and experimental gene #6: LPR-4 (Wormbase 2017). The second experimental plate contains the gene LPR-4, which encodes for the family of LiPocalin proteins (Wormbase 2017). LPR-4 is essential for transport and body muscle cell structure so if that specific gene were to be silenced, it would cause yolk endocytosis to become defective, which is necessary in nutrient transport and cellular homeostasis (Balklava et al. 2007)). The RNAi plates, with between 3-12 worms on each plate, are then incubated at 15°C for seven days (Macmillan 2017). Since negative control means that dish of worms is not exposed to experimental treatment, ie fed on bacteria without RNAi, and therefore expected to have no effect on chemotaxis (Macmillan 2017).  Worms would be attracted to diacetyl, DA side, and repelled to sodium azide, the O side of plate. The positive control subject group are fed E.Coli with ODR-10 RNAi, exposed to experimental treatment with a known effect. Worms would not be able to sense DA.