Student project

Cellular transfection of small interfering ribonucleic acids. Electroporation as an alternative to liposome-based delivery transfection methods

RNA interference has become an increasingly important tool for all aspects of molecular biology. Nevertheless, there is a technological challenge for performing gene knock-down with chemical transfection agents, as many relevant cell types are refractory to efficient transport of short interfering RNA (siRNA) into cytosol using these classical carrier-based methods. The aim of this project was to evaluate the potential of electroporation as a generic transfection method and to compare it with a standard and well-established chemical reversetransfection using cationic lipids as carriers for short-interfering RNAs. We have used an automated cell electroporation instrument from a swedish company, Cellectricon. The Cellaxess HT instrument is able to perform electroporation in 384 wells plates. We compared cell viability, transfection efficiency and extend of gene silencing caused by electroporation, with reverse-transfection using Lipofectamine RNAiMAX. Gene knock down efficiency has been measured at the protein function level by targeting ubiquitously expressed genes that are essential to cell survival. Cell viability and transfection efficiency are, then, opposed with this strategy. Results clearly showed a net improvement of "difficult-to-transfect" cell transfection but this improvement comes at the price of consuming up to ten fold more siRNAs. For "normal" cell line, lipofection remains the preferred methodology as it is more efficient and less expensive. Furthermore, the Cellaxess HT instrument has some serious drawbacks that pervert easy and reproducible result acquisition.


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