International Mammalian Genome Society

The 16th International Mouse Genome Conference (2002)


POSTER 125 - SiRNA – DESIGN, EXECUTION, AND ANALYSIS

A Goodwin
Ambion, Inc.

Brown D, Ford L, Jarvis R, Byrom M, Pallotta V, Khoeunh T
Ambion, Inc

Recent reports indicate that direct transfer of 21–23 base small interfering RNAs (siRNAs) into mammalian cells can effectively target specific RNAs for gene silencing. Here siRNA efficacy, mechanism of action, distribution, induction, and duration of action were addressed. In vitro transcribed siRNAs to mammalian genes including GAPDH, (beta)-actin, cyclophilin, c-myc, c-jun, c-fos, survivin, and the RNA-binding protein, La, had a 20X increased potency over chemically synthesized siRNAs for suppression of target expression. siRNAs were localized to the nuclear periphery and in vivo siRNA strand separation was observed. Both mRNA and protein levels remained suppressed as long as 10 days post transfection. The Silencer(TM) siRNA Construction Kit provides a cost-effective alternative to chemically synthesized siRNAs, yielding molecules of higher potency. Transfection agents optimized to deliver siRNA to a wide variety of mammalian cells at high efficiency, siPORT Amine (a polyamine mixture) and siPORT Lipid (amixture of cationic and neutral lipids), will also be described. Ambion’s Silencer(TM) siRNA Labeling Kit can append either Cy3 or FAM (Fluorescein) to siRNAs. Finally, use of plasmid vectors showed stable expression of siRNAs enabling long-term knockdown analyses. Based on these experiments, we will provide an overview of the critical issues for design, synthesis, transfection and tracking of siRNAs in mammalian cells.


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