Standard methods were used for culturing C. elegans on NGM (nematode growth medium) [30]. Fragments designated for RNAi were obtained by polymerase chain reaction (PCR) from genomic DNA and were cloned into the L4440 feeding vector (pPD129.36) [4]; all fragments were between 500 and 2,700 base pairs (bp) in length. The resulting plasmids were transformed into the HT115(DE3) RNase Ill-deficient E. coli strain, which was previously shown by Timmons and Fire to be beneficial for RNAi by feeding (L. Timmons and A. Fire, personal communication). The HT115 genotype is as follows: (F-, mcrA, mcrB, IN(rrnD-rrnE)1, lambda-, rnc14::Tn10(DE3 lysogen:lacUV5 promoter-T7 polymerase)). The RNase III gene is disrupted by a Tn10 transposon carrying a tetracycline-resistance marker. Inclusion of tetracycline in feeding plates or in bacterial cultures used for feeding in many cases resulted in a weaker RNAi effect (data not shown), perhaps because the cultures grew very poorly, and thus it was not included in our feeding experiments; however, bacteria were selected on tetracycline plates before feeding.
