standard Eating dsRNA-Expressing Bacteria to Silence Genes

Daphnia pulex

Daphnia pulex Image:Paul Herbert

Schumpert, Dudycha and Patel (2015) report efficient gene silencing in Daphnia melanica following feeding on dsRNA-expressing E. coli. This work was published recently in BMC Biotechnology.

Finding convenient modes of delivery of gene silencing RNAs has been ongoing since the emergence of RNAi as a functional genomics tool. Feeding on dsRNA directly can be quite effective in some insects and, of course, has been used as a plant protection strategy.

Aquatic arthropods lend themselves to feeding-and soaking-based gene silencing strategies and there have been a number of reports demonstrating successful variations on this theme. Chitosan has been used as a carrier of dsRNA and can be fed to aquatic arthropods. Schumpert et al. report the use of E. coli expressing dsRNA to silence genes in the microcrustacean Daphnia melanica. Similar findings have been reported in insects and shrimp

Various RNAi triggers

Various RNAi triggers

The authors targeted the phenoloxidase gene and expected reduced expression to result in less melanin and, consequently, less pigmentation. This is indeed what they observed.

Double-stranded RNA was produced in E. coli that had been transformed with plasmids containing target sequences consisting of three different regions of the phenoloxidase gene flanked on the 3’ and 5’ ends by T7 promoters. Induction of the T7 promoters resulted in dsRNA corresponding to the cloned regions. These bacteria were fed at various concentrations to D. melanica over the course of a number of days and lethality and pigmentation levels were scored.

Daphnia

Effects of gene silencing on melanization following UV exposure. WT, EV and GFP are controls and P1-3 have phenoloxidase silenced. This is part of Figure 3 from Schumpert et al. (2015). See their paper for details.

In addition to recording a visible phenotype following feeding the authors were also able to detect reductions in the levels of phenoloxidase mRNA as well as melanin content. These data show that feeding Daphnia E. coli that were expressing dsRNA can be effective at modulating gene expression.

There are some technical advantages of using E. coli, not the least of which is avoiding in vitro synthesis of dsRNA, which tends to be a bit inefficient and relatively expensive when done using commercially available kits. So it will be interesting to see if this technology will be more widely used in insect systems.

Check out this paper for more of the details. The authors note in the Discussion section that their efforts to target other genes were also successful to some extent, suggesting that this method may be generally useful.

Schumpert, C., Dudycha, J., Patel, R., 2015 Development of an efficient RNA interference method by feeding for the microcrustacean Daphnia. BMC Biotechnol 15: 91 doi:

 

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