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RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. The mechanism was discovered by Andrew Fire and Craig Mello in 1998 in plants. Today, it is observed in almost all eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. This precise cellular mechanism of gene silencing has been developed into a technique that provides an efficient way to identify and determine the...

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Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Published on: March 25, 2015

Protocols for gene silencing in schistosomes.

David Ndegwa¹, Greice Krautz-Peterson, Patrick J Skelly

¹Molecular Helminthology Laboratory, Division of Infectious Diseases, Department of Biomedical Sciences, Tufts University, Cummings School of Veterinary Medicine, Grafton, MA 01536, USA.

Experimental Parasitology

|September 18, 2007

Summary

RNA interference effectively suppresses the Schistosoma mansoni alkaline phosphatase gene in parasites. This method, using double-stranded RNA (dsRNA), shows promise for functional genomics in schistosomes.

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Area of Science:

Parasitology
Molecular Biology
Genomics

Background:

Schistosomes are platyhelminths causing widespread human infection.
Advances in schistosome genomics and molecular tools are ongoing.
RNA interference (RNAi) is a powerful gene silencing technique.

Purpose of the Study:

Develop effective RNA interference (RNAi) protocols for Schistosoma mansoni intra-mammalian stages.
Target the alkaline phosphatase gene (SmAP) using RNAi.
Establish robust gene suppression methods for schistosome functional genomics.

Main Methods:

Utilized RNA interference (RNAi) with long dsRNA and short interfering RNAs (siRNAs).
Targeted the Schistosoma mansoni alkaline phosphatase (SmAP) gene.
Employed electroporation for dsRNA delivery to cultured schistosomula and adult schistosomes.
Quantified SmAP RNA levels using quantitative real-time PCR.
Measured alkaline phosphatase enzyme activity.

Main Results:

Both long dsRNA and siRNAs effectively suppressed SmAP gene expression.
Electroporation proved more efficient for dsRNA delivery than soaking.
SmAP RNA levels decreased by over 90% within 2 days post-electroporation.
Alkaline phosphatase enzyme activity decreased by over 70% within 5 days post-treatment.

Conclusions:

Developed and validated effective RNA interference (RNAi) protocols for Schistosoma mansoni.
Demonstrated robust gene suppression in intra-mammalian schistosome stages.
These protocols have wide applicability for functional schistosome genomics and parasite research.