Development of an in Vitro RNAi to Investigate Gene Function in Brugia Malayi

Abstract

The RNA interference (RNAi) has the ability to turn off individual gene expression. So, it affords a remarkably specific tool for studying the effects of genes. It is regarded as a direct approach for determining such gene/genes functions and offers a valuable tool for modern drug discovery. The study aimed to develop in vitro RNAil in Brugia malayi with particular interest to study the function of Brugia malayi avr-14 (Bm-avr-14) and Brugia malayi f-tubulin (Bm-fi-tubulin) genes. Bm-avr-14 is a gene encoding glutamate gated chloride channel (GluCl) which binds ivermectin and Bm-β-tubulin is a gene encoding β-tubulin which binds albendazole. Adult female worms were soaked in heterogeneous short interfering RNA (hsiRNA) with interest to study the role of two genes Bm-avr-14 and Bm-β-tubulin. Then, we assessed the knock down effects of target genes using worminator system and real time PCR. We found that worms treated with hsiRNA of Bm-avr-14 had a significant reduction in microfilariae (mf) production in comparison with untreated worms or worms treated with hsiRNA of green fluorescent protein (GFP). No significant reduction in mf production with Bm-β-tubulin gene was obtained. There were no changes in the movement of adults treated with either Bm-avr-14 or Bm- β-tubulin hsiRNAs. Inconsistent RNAi mediated suppression was achieved with Bm-avr-14 and Bm-β- tubulin using real time PCR. The data may be helpful in assessment of drug target potential of genes.