( iver siRNAs into i...)Delivering siRNAs to Difficult Cell Types

iver siRNAs into immortalized cells. Unfortunately, efficient transfer of siRNAs into primary cells by chemical transfection is restricted to only a few cell types [1]. Since primary cells are more similar to their in vivo counterparts than are immortalized cells, they serve as an important model system for in vivo applications. Better delivery methods for these cell types are needed.

As an alternative to chemical transfection, Ambion has investigated electroporation of primary cells and hard-to-transfect cell types. Electroporation involves applying an electric field pulse to induce the formation of microscopic pores in the cell membrane which allow molecules, ions, and water to traverse the membrane [2]. Under specific pulse conditions, the pores reseal and the "electroporated" cells recover and resume growth. A distinct advantage of electroporation over chemical methods is that it is not dependent on cell division, and RNAi-induced reduction in gene expression can be detected just a few hours after nucleic acid delivery.

Most existing electroporation protocols were developed to deliver plasmid DNA to the cell nucleus [3, 4]. These protocols often result in high cell mortality [5, 6, 7]. For transient RNAi experiments, it is important that cell trauma from electroporation be minimized to reduce any possible effects on gene expression patterns. Since siRNAs need only be delivered to the cytoplasm, milder electroporation conditions can be used that minimize cellular mortalit
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