Investigations on the lgx-1 gene in Caenorhabditis elegans.
Yen, Lawrence L.
- Lymphatic filariasis affects around 120 million people world wide (WHO), and is caused by nematodes Brugia malayi, Brugia timori, and Wuchereria bancrofti in many developing countries (Duke, 2001). The chitin synthesis and processing pathway may serve as a potential drug target for filariasis as the nematodes that cause the disease produce chitin while vertebrates do not. The model organism chosen... read morefor the studies presented in this paper is Caenorhabditis elegans (C. elegans). C. elegans shares the chitin synthesis and processing pathways with other nematodes and its entire genome has previously been sequenced. The lgx-1 gene in C. elegans codes for a putative chitin deacetylase and has been shown to be expressed in the pharynx of the organism by in situ hybridization experiments (Kohara, 2010). We therefore hypothesize that knocking down the lgx-1 gene in C. elegans would affect the function of the pharynx. For this study, we attempted to produce dsRNA of the chitin deacetylase (CDA) region of the lgx-1 gene in vitro to be used for RNAi experiments by soaking. The in vitro transcription reactions using a short cDNA fragment representing the CDA domain resulted in no sizeable yield of full length transcripts. We also cloned a larger cDNA fragment containing exons 12 -21 of the lgx-1 gene, known as the yk1621B03 construct, into the Litmus 28i vector. As a final part of this study, we performed an RNAi experiment by feeding C. elegans with bacteria expressing dsRNA from two different lgx-1 cDNA fragments. The results showed no significant difference in brood size between the experimental and control populations and an unexpected larger population of nematodes in the experimental samples developing into the L3, L4, and adult stages relative to the control samples.read less