Transgenic RNAi Using GAL4/UAS System in Bombyx mori

Abstract

RNA interference (RNAi) is an important tool to suppress the gene expression in a eukaryote. To examine a utility of transgenic RNAi in the silkworm Bombyx mori, we constructed the piggyBac-based RNAi vector, which was designed to produce hairpin double-stranded RNA (hairpin-dsRNA) under the control of the GAL4/UAS system. First, we determined the effectiveness of this vector to suppress the ectopic expression of firefly luciferase in cultured cells. The results showed that the luciferase activity in the cells that were transfected with the expression plasmids was markedly suppressed by producing hairpin-dsRNA. Then, we generated transgenic silkworms producing hairpin-dsRNA corresponding to the endogenous gene such as w-3, fibroin heavy chain (fib-H), and sericin1 (ser1) genes using a binary GAL4/UAS expression system. The analysis of the transgenic silkworms showed that the amounts of their transcripts were significantly decreased in the expression of the hairpin-dsRNA. The translucent integument was observed in the transgenic lines producing hairpin-dsRNA of w-3, and the weight of cocoon layer decreased in the transgenic lines producing hairpin-dsRNA of fib-H or ser1. These results suggest that transgenic RNAi under the control of the GAL4/UAS system is very effective on gene knockdown at the larval stage and a useful tool to analyze gene functions of the silkworm.