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.

On the generality of RNA-mediated interference

Andrew Fire, Jamie Fleenor

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.