Two of our methods are now seeing publication. We are writing to let people know how to get the relevant strains and constructs. The first method uses the ninth intron of mec-2, whose splicing depends on mec-8, to produce temperature-sensitive expression and temperature-sensitive RNAi (Calixto et al., 2010a). The intron 9 vector TU#821 (Pmec-18 intron-9 mec-2::yfp) is available from Addgene (www.addgene.org/Martin_Chalfie). The TU218 [mec-8(u218ts)] and TU3135 [mec-8(u218); rde-1(ne219); uIs46 (ceh-22::GFP, Prde-1mec-2 intron9::rde-1)] strains are available from the C. elegans Genetic Center (CGC).
The second method uses SID-1 expression in neurons to allow feeding RNAi for neuronally-expressed genes (Calixto et al., 2010b). Expression of neuronal sid-1 in the RNAi sensitized strain lin-15b(n744) greatly enhanced this effect. Furthermore, promoter-driven sid-1(+) can be used with a sid-1 mutant background to restrict RNAi to specific cells. Currently we are using neuronal and touch receptor neuron-specific expression of sid-1 to uncover neuronal and touch neuron-specific defects for genes whose loss has more general effects (e.g. lethality). The following strains have been submitted to the CGC. In addition, a vector allowing pan-neuronal sid-1 expression TU#867 (Punc-119 sid-1) will be available from Addgene.
|TU3311||uIs60 (Punc-119yfp, Punc-119sid-1)|
|TU3335||uIs57 (Punc-119yfp, Punc-119sid-1, Pmec-6mec-6); lin-15b(n744)|
|TU3401||uIs69 [pCFJ90 (Pmyo-2mCherry), Punc-119sid-1]; sid-1(pk3321)|
|TU3595||uIs72 [pCFJ90 (Pmyo-2mCherry), Punc-119sid-1, Pmec-18mec-18::gfp]; sid-1(pk3321) him-5(e1490); lin-15b(n744)|
|TU3568||uIs71 [pCFJ90 (Pmyo-2mCherry), Pmec-18sid-1]; sid-1(pk3321) him-5(e1490); lin-15b(n744)|
Calixto A, Ma C, Chalfie M. (2010a). Conditional gene expression and RNAi using MEC-8-dependent splicing in C. elegans. Nat. Methods 7, 407-411.
Calixto A, Chelur D, Topalidou I, Chen X, Chalfie M. (2010b). Enhanced neuronal RNAi in C. elegansusing SID-1, Nat. Methods, [Epub ahead of print].
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