Worm Breeder's Gazette 15(3): 8 (June 1, 1998)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Carnegie Institution of Washington, Department of Embryology, 115 West University Parkway, Baltimore, Md. 21210 USA
After suggestions from Mei Hsu and from Mike Nonet that some tissues might show reduced sensitivity to RNA-mediated interference (RNAi), we have investigated the generality of RNAi using a broadly expressed gfp-reporter transgene. As a target for these experiments, we needed a gfp fusion that was expressed in all somatic tissues. For this purpose, we fused the upstream region from the ribosomal protein L5 gene to gfp (with a nucleolar localization signal to facilitate cell identification). A transgenic line with this fusion expressed nucleolar GFP in all somatic tissues. Although GFP was stronger in younger larvae than in adults, some activity was retained in all cells throughout the life of the animals. Activity was comparable in different cell types (gut showed slightly more activity and neurons slightly less, with muscle and hypodermis intermediate, but these differences were not extreme). We injected young adult animals from this strain with a double-stranded RNA hairpin corresponding to the complete coding sequence for gfp. RNA was injected at a high concentration (~1mg/ml) to obtain maximal interference, and animals were then grown at 25!C under standard conditions. We then examined gfp activity patterns in the injected animals and in their progeny. *Injected animals ("I0"): Within three days, GFP was essentially eliminated in all muscle, somatic gonad, hypodermal, and intestinal cells. Staining in the nervous system appeared to decrease somewhat but was not fully eliminated: a high fraction of neurons (particularly in the head or tail) contained GFP even after the RNA injection. Control animals (uninjected) retained activity in all tissues. *Progeny Animals ("F1"): In young and mid-stage larvae, GFP was absent in the vast majority of muscle, hypodermal, and intestinal cells (although sporadic cells of these classes were GFP+). By contrast, a high fraction of cells in the nervous system retained GFP activity. We assume that these neuronal cells were at least partially able to resist or escape the the RNAi. This was particularly true for cells in the head and tail region, while the ventral cord motor-neurons were frequently affected by the RNAi. Even in the head and tail, some nervous system cells are clearly affected (GFP-) in each animal, while others continued to express at least a fraction of the GFP level seen in uninjected controls. It is not clear whether the general pattern of RNAi-resistant and RNAi-sensitive neuronal cells varies from animal to animal or is constant in the population. *F2 animals: All F2 animals that we examined showed a return to the fully GFP+ phenotype of the original strain.