siRNA-directed silencing of transgene expressed in cultured insect cells.
RNA interference (RNAi) has emerged as a powerful tool to rapidly analyze gene functions in a wide variety of eukaryotic organisms as well as in cultured cell lines. We demonstrate here that RNAi can be applied to study the function of a transgene expressed in an insect cell line (Spodoptera frugiperda, Sf21). The aminopeptidase N gene (apn) targeted for silencing in the present study was isolated from the midgut of Spodoptera litura larvae and expressed in Sf21 cells using baculovirus expression system. The recombinant APN protein expressed at the surface of Sf21 cells was shown to interact with insecticidal crystal protein, Cry1C, by in vitro experiments. The exogenous addition/transfection of APN dsRNA or siRNA in the cultured cells resulted in partial/complete inhibition of expression of apn leading to the loss of toxin binding to the transgene expressing cells. These experiments highlighted the usefulness of RNAi as a tool to study the function of an expressed transgene in insect cell line and to study the specificity of receptor-ligand interaction.
Agrawal
Neema
N
International Center for Genetic Engineering and Biotechnology, New Delhi 110 067, India.
Malhotra
Pawan
P
Bhatnagar
Raj K
RK
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002478:Cells, Cultured; D020868:Gene Silencing / Q000502:physiology*; D008024:Ligands; D012313:RNA; D034741:RNA, Small Interfering / Q000502:physiology*; D018411:Spodoptera / Q000166:cytology / Q000235:genetics*; D019076:Transgenes
D008024:Ligands; C034477:RNA primers; D034741:RNA, Small Interfering; D012313:RNA