A universal transgene silencing approach in baculovirus-insect cell system.
Baculovirus-insect cell system (BICS) is considered one of the most efficient eukaryotic gene expression systems. This system has also been used for producing different recombinant baculoviruses with increased insect toxicity as potential biopesticides. Establishing a universal gene silencing (UGS) system is very important due to the increasing number of studies using RNA interference (RNAi) with BICS. In this work, the enhanced green fluorescent protein (EGFP) was used as the RNAi consistent target sequence located downstream of a depressant insect-neurotoxin gene, LqqIT2 used as a model of the gene of interest. Small interfering RNA (siRNA) and inverted repeats of EGFP gene (IR-EG) were examined in targeting the EGFP-LqqIT2 (EL)-fusion mRNA or LqqIT2-EGFP (LE)-bicistronic mRNA for degradation. Suppression efficiencies using these inducers were examined transiently and stably in uninfected and infected insect Sf9 cells. Moreover, RNAi showed persistence for 4 and 8 days in baculovirus-infected as well as uninfected Sf9 cells, respectively. Bicistronic RNA seems an efficient way to lower cost and effort of the gene silencing approach while maintaining the biological activity of the protein of interest when the RNAi is not in use.
Salem
Tamer Z
TZ
Department of Microbiology and Cell Science, University of Florida, P.O. Box 110700, Gainesville, FL 32611, USA. zakiti@muohio.edu
Maruniak
James E
JE
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D018411:Spodoptera; D014162:Transfection; D019076:Transgenes
D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins