Description
TitleNovel lifespan and RNA interference assays for Caenorhabditis elegans
Date Created2022
Other Date2022-05 (degree)
Extent159 pages : illustrations
DescriptionThe nematode C. elegans has experienced rapid growth as a scientific model system since its first usage by Sidney Brenner and other scientists in the 1960’s. At the time of this publication, studies in C. elegans have generated six Nobel prizes, to Sidney Brenner, John Sulston, Robert Horovitz, Martin Chalfie, Andrew Fire, and Craig Mello. This is a remarkable number of awards considering the short lifetime of the C. elegans research field. The discovery of programmed cell death, RNA interference (RNAi), green fluorescent protein, the exopher, the first whole genome sequence in a multicellular organism, and the first complete connectome, are just some of the major accomplishments that can be attributed to the C. elegans model system. However, as a relatively newer and niche avenue of study, many novel technologies that are being developed in science do not focus on applications for nematode research. Consequently, many assays currently performed in the C. elegans model still require considerable manual labor, which is particularly unfortunate as C. elegans is uniquely suited for a variety of high throughput assays due to its easily manipulatable genetics, low cost, short lifespan and hermaphroditism. Two common C. elegans assays, lifespans and RNAi screening, both require considerable manual labor. RNAi screens are performed by growing and plating individual bacterial clones from a full genome RNAi library. Worm growth is then synchronized prior to animal transfer onto RNAi plates and scoring. Numerous efforts have been made to automate these assays. The goal of my dissertation was to make RNAi and lifespan assays accessible at a scale that takes advantage of the possibilities of automation. One of the best automated lifespan methods uses the Lifespan Machine, which is a flatbed scanner device that collects images and features software that automatically tracks animals until their death. The Lifespan Machine massively increases lifespan screen capacity, allowing these large experiments to be performed at a scale not possible using manual approaches. However, instrument construction is relatively complicated, which limits widespread adoption of this technology in the field. My goal was to simplify the design to the point that anyone could setup a Lifespan Machine with minimal effort and resources; an effort that I published as a chapter in this thesis. The novel assay technologies I designed have already borne fruit. The simplified lifespan machine design is being used by the C. elegans Intervention Testing Program, a multi-institute project that is investigating potential drugs for improvements in lifespan or healthspan.
C. elegans RNAi screens can enable the rapid identification of genes affecting myriad biological processes. RNAi screens have been automated, but the best attempts currently still require manual pipetting, repeated plate transfers, and use of liquid culture that can alter nematode physiology. I developed a fully automated RNAi screen protocol that rivals RNAi screens done in unicellular organisms for speed and ease of performance without the use of prohibitively expensive fully robotic facilities. I validated the RNAi screening assay using the cysl-2 stress response pathway, with data both confirming known genes from the literature and identifying novel genes of interest that may play a role in C. elegans cysl-2 biology. There are many important mechanistic questions to be addressed in C. elegans. One process of interest in the Driscoll lab involves a novel extrusion vesicle called an “exopher” that may play a conserved role in protein aggregation diseases. The exopher pathway is currently completely uncharacterized, and therefore a whole genome RNAi screen to identify genes required for exopher production would likely pave new ground in the field. Exophers, which can be relatively rare and difficult to distinguish from cell somas, present complex challenges for a screen, and my work on a whole genome screen for exopher modulator genes has reached an exciting phase. I have completed strain development and initiated the screen, with preliminary observations reported herein.
NotePh.D.
NoteIncludes bibliographical references
Genretheses
LanguageEnglish
CollectionSchool of Graduate Studies Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.
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