siRNA-directed silencing of transgene expressed in cultured insect cells

Biochemical and Biophysical Research Communications
Neema AgrawalRaj K Bhatnagar

Abstract

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.

References

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Citations

Sep 17, 2004·Applied Microbiology and Biotechnology·S JanaJ K Deb
Mar 24, 2006·Biotechnology Letters·Chih-Chien LinYiu-Kay Lai
Dec 29, 2009·Journal of Insect Physiology·Chenxi LiuYuyuan Guo
Jul 8, 2008·Journal of Virological Methods·Sally HiltonDoreen Winstanley
Jun 6, 2007·Journal of Virological Methods·Tamer Z Salem, James E Maruniak
Aug 2, 2011·Biotechnology Advances·Varnika BhatiaRamcharan Bhattacharya
Jan 19, 2008·Orvosi hetilap·Zsuzsanna Rácz, Péter Hamar
Feb 6, 2009·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Gatikrushna SinghRaj K Bhatnagar
Mar 12, 2017·Applied Biochemistry and Biotechnology·Naglaa A AbdallahAbdelhadi Abdallah Abdelhadi

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