( ers of the RNAi effect i...)RNAi in Non-Mammalian Cultured Cells

ers of the RNAi effect in cultured Drosophila melanogaster cells. The MEGAscript RNAi Kit was used to generate full-length dsRNA to the Drosophila hrp48 and U2AF⁵⁰ genes from the corresponding cDNA constructs. As no transfection is required for insect cells, 1x10⁶ Schneider's Drosophila L2 cells were grown in 6 well plates in serum free medium and directly treated with 10 nM of dsRNA. Western blot analysis 72 hours after treatment showed that hrp48 and U2AF⁵⁰ protein expression levels were reduced by 98% and 80%, respectively (Figure 2A). The silencing effect was specific and dose-dependent (Figure 2A and 2C). A strong correlation between suppression of mRNA and protein expression was demonstrated by Northern blot analysis (Figure 2A and 2B). This observation supports the model that dsRNA-triggered gene silencing results from a reduction in the amount of target mRNA available for translation.

Figure 2. dsRNA-Mediated Silencing of Drosophila U2AF⁵⁰ and hrp48 Gene Expression. ( A) Specific reduction of protein expression levels. The RNAi effect was analyzed by Western blot with anti-U2AF⁵⁰ (50 kDa) and anti-hrp48 (48 kDa; migrates as a doublet) antibodies. An antibody directed against Pab 1 (lower panel) was used as an additional negative control. (B) Specific reduction of mRNA expression levels. Total RNA samples were purified (RNAqueous Kit, Ambion) and analyzed using by Northern blotting ('"/>

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