Summary
RNA-mediated interference (RNAi) has been a valuable tool for the analysis of gene function in Caenorhabditis elegans (C. elegans). In C. elegans, the injection of double-stranded RNA (dsRNA) or plasmid DNA expressing dsRNA under the control of a C. elegans promoter results in gene inactivation through the specific degradation of the targeted endogeneous mRNA. It is also possible to initiate RNAi either by soaking worms in a solution of dsRNA or by feeding worms with E. coli expressing the dsRNA. Using studies of the DNA repair in C. elegans as an example, we describe the use of RNAi against the C. elegans POLH gene (Ce-POLH), which encodes DNA polymerase η (pol η).
Pol η has the ability to catalyze translesion synthesis (TLS) past UV-induced cyclobutane pyrimidine dimers (CPDs) and some other lesions as well. Loss of pol η in humans results in increased photosensitivity and the cancer-prone genetic disorder xeroderma pigmentosum variant (XPV). We provide an example of the feeding RNAi technique, in which downregulation of pol η in C. elegans results in increased sensitivity of several development and differentiation processes, including meiosis and embryogenesis to UV radiation.
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Acknowledgments
We thank Dr. Hideyuki Okano for providing the C. elegans embryonic cDNA library and Dr. Andrew Fire for providing the bacterial strain HT115 (DE3) and the vector L4440 (pPD129.36). We are grateful to Dr. Yoshiaki Ohkuma, Dr. Masayuki Yokoi, and other members of Dr. Hanaoka's laboratory at Osaka University for helpful discussions. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by the Human Frontier Science Program, and by Solution Oriented Research for Science and Technology (SORST) from the Japan Science and Technology Agency.
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Ohkumo, T., Masutani, C., Eki, T., Hanaoka, F. (2008). Use of RNAi in C. elegans . In: Barik, S. (eds) RNAi. Methods in Molecular Biology™, vol 442. Humana Press. https://doi.org/10.1007/978-1-59745-191-8_10
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DOI: https://doi.org/10.1007/978-1-59745-191-8_10
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