siRNA-directed silencing of transgene expressed in cultured insect cells

Agrawal, Neema ; Malhotra, Pawan ; Bhatnagar, Raj K. (2004) siRNA-directed silencing of transgene expressed in cultured insect cells Biochemical and Biophysical Research Communications, 320 (2). pp. 428-434. ISSN 0006-291X

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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.

Item Type:Article
Source:Copyright of this article belongs to Elsevier Science.
Keywords:RNA Interference; Baculovirus Expression System; Insect Cells; Small Interfering RNA; Double-Stranded RNA; Protein–Protein Interaction
ID Code:103471
Deposited On:01 Feb 2018 17:35
Last Modified:01 Feb 2018 17:35

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