RNAi Plating voor C. elegans Feeding: Een techniek om doel dsRNA expressie te induceren in E. coli

- In C. elegans, RNAi can be induced by feeding worms bacteria that express double-stranded RNA or dsRNA from a recombinant DNA plasmid.

To begin, grow tetracycline-resistant HT115 E. coli transfected with L4440 plasmid overnight on selective LB agar plates containing tetracycline and ampicillin, or its functional analog carbenicillin. In addition to the target DNA, the plasmid contains a gene for ampicillin resistance. Only bacteria colonies that inherit the plasmid can grow in the presence of these antibiotics.

Next, harvest bacteria colonies and expand them in liquid LB medium to the desired concentration. To induce dsRNA expression, transfer the bacteria solution onto prepared nematode growth medium, or NGM, agar plates containing IPTG.

In this expression system, IPTG mimics lactose to inactivate the lac repressor enabling the expression of T7 RNA polymerase. On the plasmid, target DNA expression is regulated by two convergent T7 promoters. Consequently, sense and anti-sense sequences are transcribed leading to the expression of dsRNA.

Finally, the worm uses the sequence information from the ingested dsRNA to downregulate endogenous mRNAs with complementary sequences. In this e