Application of simple and massive purification system of dsRNA in vivo for acute toxicity to Daphnia magna
Choi, W., National Institute of Ecology, Seocheon, Republic of Korea | Lim, H.S., National Institute of Ecology, Seocheon, Republic of Korea | Kim, J., Central Research Institute, Kyungnong Co., Gyeongju, Republic of Korea | Ryu, S.M., Central Research Institute, Kyungnong Co., Gyeongju, Republic of Korea | Lee, J.R., National Institute of Ecology, Seocheon, Republic of Korea
The RNA interference (RNAi) has been considered as an important genetic tool andapplied to develop a new living modified (LM) crop trait which is an improvement ofnutrient quality or pest management. The RNAi of DvSnf7 has been used forresistance to LM maize and the Western Corn Rootworm which is a majoragricultural pest for the US Corn Belt. Most of the environmental risk assessments(ERA) of double strand RNA (dsRNA) have been performed usingin vitrotranscriptproducts, and notin vivoexpressed product. A large amount of dsRNA was requiredfor the acute toxicity assay of water fleas. Therefore development of massive dsRNApurification techniques is critical. Daphnia, a freshwater microcrustacean, is a modelorganism for studying cellular and molecular mechanism involved in life historytraits and ecotoxicology. In this study, we established the massive dsRNApurification method usingEscherichia coliand implemented acute toxicity assaystoDaphnia magna. As a result, the present RNase A and DNase I, dsRNA wasefficiently purified without any special techniques or equipment. Even thoughpurified dsRNA existed during the acute toxicity test, lethality or abnormal behaviorwere not observed inD. magna. These results indicated that GFP and DvSnf7dsRNA were not significantly affected toD. magnadue to their lack of sequencematching in its genome. The purification method of dsRNA and the acute toxicityassay of water fleas using purified dsRNA would be suitable for the toxicologicalstudies of LMOs to aquatic non-target organisms.
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