Project description:We identified target genes for NHR-25 by ChIP-seq at L1 stage of C. elegans. Transcription factor genes were tagged with GFP and their expression examined at L1 stage. Since there are no direct target genes known for NHR-25 that can be used for assessment of enrichment efficiency by quantitative PCR (qPCR), we chose to repeat ChIP-seq experiment of another GFP tagged transcription factor, PHA-4 for which the ChIP-seq was performed during a pilot experiment of modENCODE project using the same transgenic strain and antibody (a gift from Tony Hyman lab). pha-4 and nhr-25 transgenic worm were studied in Fed L1 stage.
Project description:The gene nhr-6 encodes the sole C. elegans NR4A nuclear receptor homolog which has a critical role in organogenesis by regulating the development of the spermatheca organ. Here we utilized chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) to identify NHR-6 binding sites during both the late L3/early L4 and mid L4 developmental stages. Our results revealed 30,745 enriched binding sites for NHR-6 with sites specific to each developmental stage and sites found in both stages. Additionally, ~70% of enriched sites were found within 3 kb upstream of a gene transcription start site. Binding sites for a cohort of candidate target genes with probable functions in spermatheca organogenesis were validated through qPCR. Reproductive and spermatheca phenotypes were also evaluated for these genes following a loss-of-function RNAi screen which revealed several genes with critical functions during spermatheca organogenesis. Our results uncovered a complex nuclear receptor regulatory network whereby NHR-6 regulates multiple cellular processes including proliferation, differentiation, and metabolism.
Project description:We identified target genes for NHR-25 by ChIP-seq at L1 stage of C. elegans. Transcription factor genes were tagged with GFP and their expression examined at L1 stage. Since there are no direct target genes known for NHR-25 that can be used for assessment of enrichment efficiency by quantitative PCR (qPCR), we chose to repeat ChIP-seq experiment of another GFP tagged transcription factor, PHA-4 for which the ChIP-seq was performed during a pilot experiment of modENCODE project using the same transgenic strain and antibody (a gift from Tony Hyman lab).
Project description:Purpose: Naringin (Nar) is a dihydroflavonoid compound, which is widely found in Chinese herbal medicine and citrus fruit. As one of the phytochemicals, it acts as a dietary supplement and can delay aging and prevent aging-related disease, such as obesity and diabetes. However, the exact mechanism still partially remained unclear. Methods: In this study, the high-glucose induced (HGI) Caenorhabditis elegans model was used to evaluate the anti-aging and anti-obesity effects of Nar. Results: It indicated that the mean lifespan and fast movement span of HGI worms were extended roughly 24% and 11% by Nar treatment, respectively. Oil red O staining revealed the significant reduction of fat accumulation (P<0.05) and the dFP::LGG labeled worms showed the promotion of autophagy. Additionally, the whole transcript sequencing and gene set variation analysis algorithm suggested that Nar improved the lipid biosynthesis and metabolism pathways, as well as the TGF-β, Wnt and longevity signaling pathway. The PPI network was applied to screen out hub genes in the above-mentioned pathways for further analysis. The expression levels of hub genes fat-7 and ech-6 were down-regulated, as well as aak-2, nhr-49, acs-2, hlh-30, lgg-1, unc-51, pha-4, skn-1 and yap-1 were up-regulated. Then, mutant worms and RNA interference were used to study the mechanism. It revealed that suppression of the expression of autophagy related genes including hlh-30, lgg-1, unc-51, pha-4, skn-1 and yap-1 could disable the fat-lowering, lifespan-prolonging, and health-promoting properties of Nar. Finally, a competing endogenous RNAs network was constructed to profoundly understand the beneficial mechanism caused by Nar. Conclusions: our findings elucidate that Nar plays the important role to alleviate HGI aging and anti-obesity effects by reducing fat accumulation and promoting autophagy.
Project description:Microinjection is the most frequently used tool for genetic transformation of the nematode Caenorhabditis elegans, facilitating the transgenic expression of genes, genome editing by the clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 system, or transcription of dsRNA for RNA intereference (RNAi). Exogenous DNA is delivered into the developing oocytes in the germline of adult hermaphrodites, which then generate transgenic animals among their offspring. In this protocol, we describe the microinjection procedure and the subsequent selection of transgenic progeny.
Project description:Caenorhabditis elegans is a free-living nematode that resides in soil and typically feeds on bacteria. We postulate that haematophagic C. elegans could provide a model to evaluate vaccine responses to intestinal proteins from hematophagous nematode parasites, such as Necator americanus. Human erythrocytes, fluorescently labelled with tetramethylrhodamine succinimidyl ester, demonstrated a stable bright emission and facilitated visualization of feeding events with fluorescent microscopy. C. elegans were observed feeding on erythrocytes and were shown to rupture red blood cells upon capture to release and ingest their contents. In addition, C. elegans survived equally on a diet of erythrocytes. There was no statistically significant difference in survival when compared with a diet of Escherichia coli OP50. The enzymes responsible for the digestion and detoxification of haem and haemoglobin, which are key components of the hookworm vaccine, were found in the C. elegans intestine. These findings support our postulate that free-living nematodes could provide a model for the assessment of neutralizing antibodies to current and future hematophagous parasite vaccine candidates.
Project description:We demonstrate for the first time the dielectrophoretic trapping and manipulation of a whole animal, the nematode Caenorhabditis elegans. We studied the effect of the electric field on the nematode as a function of field intensity and frequency. We identified a range of electric field intensities and frequencies that trap worms without apparent adverse effect on their viability. Worms tethered by dielectrophoresis (DEP) exhibit behavioral responses to blue light, indicating that at least some of the nervous system functions are unimpaired by the electrical field. DEP is useful to dynamically tether nematodes, sort nematodes according to size, and separate dead worms from live ones.