Evaluation of gene silencing in Triatoma infestans using dsRNA
Campos ITN 1; Araujo RN 2; Pereira MH 2 and Tanaka AS 1.
1Departamento de Bioquímica, UNIFESP – EPM, São Paulo, Brasil; 2 Departamento de Parasitologia, ICB, UFMG, Belo Horizonte, Minas Gerais. E-mail: ivan.bioq@epm.br
Gene silencing is a new technology which the use is growing up fast in the last years. One of the most used techniques is the RNA interference (RNAi). This technique allow us to study the function of a specific gene, eliminating the mRNA of interest by a complex of multi-proteins named RISC (RNA-induced silencing complex), which is present on cells of a vast number of eukaryotic organisms. It's proposed that this machinery is a cellular mechanism for viral protection. On the actual concern, double strands of RNA (dsRNA) are cleaved by a RNAse III related endonuclease named Dicer on the cytoplasm of the cell. The generated fragments of 21-25 bp (siRNA) induce the RISC formation which recognizes and cleaves complementary mRNA. So, according to the target cell or organism, it is possible to induce gene silencing using dsRNA, siRNA and shRNA (short hairpin RNA, a palindromic single strand of RNA that forms a hairpin).
In this work, we evaluated the use of dsRNA to induce gene silencing in the kissing bug Triatoma infestans. So, we produced 657 bases complementary RNA strand of the infestin gene (a multi domain protease inhibitor) and a not present gene (β-lactamase), hybridized and injected them into the hemolymph of 4o instar nymphs of T. infestans. Animals after two injections (10 mg dsRNA each) in an interval of 48 h were dissected; anterior midguts were used in RNA and cDNA preparations. Using semi quantitative PCR, we could detect the infestin gene in animals of the controls groups but we do not detect or barely detect the infestin gene in the infestin silenced group. Similar experiments were done using Triatoma brasiliensis and showed that the inhibitory activity present in the anterior midgut decreases significantly with infestin correlated gene silencing. Perspectives of this work are to analyze the effect of the infestin silencing in the inhibitory activity present on the anterior midgut and also analyze the effect of infestin silencing in the hematophagy. Supported by: FAPESP and CNPq
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