In vivo transient expression for the functional analysis of polydnaviral genes

Abstract

Transient expression of a foreign gene in an organism is useful to determine its physiological function. This study introduces an efficient expression technique in the insect system using a recombinant eukaryotic expression vector. A recombinant construct expressing an enhanced green fluorescence protein (EGFP) gene under an immediately early promoter was injected into the larval hemocoel of Spodoptera exigua along with a cell transfection reagent. The expression of EGFP occurred earlier, and persisted for longer period with increasing injection dose. However, there was significant variation in expression efficiency among different cell transfection reagents. In addition, the transfection efficiency measured by RT-PCR varied among tissues with high expression of EGFP in hemocytes and fat body, but not in epidermis, gut, and nerve tissues. Two functional genes (CpBV15α and CpBV15β) derived from a polydnavirus were inserted into the eukaryotic expression vector and injected into S. exigua larvae. Expression levels in hemocytes and fat body were measured by RT-PCR and immunofluorescence assay. Both mRNAs and proteins were detected in the two tissues, in which expression signals depended on the amount of injected DNA. These immunosuppressive factors significantly inhibited hemocyte behavior, such as hemocyte-spreading, nodule formation, and phagocytosis. These results demonstrate the use of in vivo transient expression of polydnaviral genes for direct analysis of biological function in the host insect.