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Moving nucleic acids into cells
Schematic comparing forward and reverse transfection.
Workflow comparison of forward transfection and reverse transfection, illustrating the labor- and reagent-saving features.
Transfection is the deliberate introduction of nucleic acids into eukaryotic cells. Common transfection examples include introducing DNA plasmids that have gene inserts for expression, or small interfering RNA (siRNA) for targeted gene silencing.
Cationic lipid-or polymer-based reagents are the most common means of transfection and suitable to most cell types. Transfection reagents couple a nucleic acid or an expression plasmid to a cationic lipid or polymer producing a liposome that interacts with the cell membrane and results in endocytosis of the molecule.
Viral-mediated transduction is a good alternative for difficult-to-transfect cell lines, with the added beneficial option of stable cell line creation. DNA within expression vectors can be packaged into viral particles, and then introduced into cells. The virus particle infects the cells and the construct is integrated into the genome, causing the internal host machinery to replicate the exogenous nucleic acids.
Electroporation is a physical means of delivery that is usually the harshest to the cells because it disrupts the cell membrane creating entry for the nucleic acid. This is a good choice for hard-to-transfect cell lines but results in the highest cell death.
In the case of siRNA delivery, a new novel technology exists, in which the siRNA is chemically modified to facilitate uptake into the cell without the need for transfection reagents, instruments, or viral vectors. Modified siRNAs achieve gene silencing in cells that had been beyond the reach of conventional RNAi methods due to toxicity from transfection reagents or undesirable viral responses.
Transfection can also occur in vivo. With live animal systems, nucleic acids are delivered systemically or locally to target tissues. In animal models there are additional factors to consider, including the presence of nucleases and the animal's innate inflammatory response, which can interfere with transfection efficiency.
Optimizing transfection protocols ensures maximal nucleic acid uptake by a majority of cells while minimizing cytotoxicity. Maximal transfection efficiency benefits the entire downstream workflow, which can encompass analysis of recombinant clones, gene expression, or gene silencing studies.
This video gives an overview of CRISPR-Cas9 gene editing, and details how the Edit-R™ CRISPR-Cas9 Gene Engineering Platform simplifies the workflow with the use of synthetic RNA.
View video (3:19)