Transfection is the introduction of any nucleic acid molecule by non-viral means into cultured eukaryotic cells. In the past, it was often used only in reference to DNA, but that has changed as applications such as RNAi and more recently, CRISPR have been developed. Although there are many ways to deliver genes to cells, there are three highly recognized methods researchers utilize today: chemical reagents, electroporation (gene electrotransfer) and viral transduction. The ultimate goal is to deliver nucleic acids into cells to investigate gene function by expression of exogenous genes or through knockdown of endogenous genes. Manipulation of gene expression is a core technique in research areas such as drug development, cancer research, gene therapy and tissue engineering.
Chemical Transfection of Eukaryotic Cells
Reagent is combined with nucleic acid to form positively charged complexes. 2) Complexes are added to cells, and bind to the negatively charged cell surfaces via electrostatic interactions. 3) Cells internalize complexes via endocytosis into membrane vesicles known as endosomes. 4) Reagent destabilizes endosomal membrane 5) Complexes escape from endosomes and release nucleic acid cargo in cytoplasm (siRNA, miRNA, or large RNA are generally active in cytoplasm). 6) DNA must localize to the nucleus, where gene expression cassette is transcribed.
Transfections using chemical transfection reagents rely on electrostatic interactions to bind with nucleic acids and to target cell membranes. This can be achieved with compounds like calcium phosphate, polycations, and liposomes or more current technologies such as cationic lipids, polymers, dendrimers, and nanoparticles.
Using calcium phosphate for delivery is the oldest and least expensive way to introduce nucleic acids into cells. This technique works well in some easy to transfect cell lines, but cannot deliver to more resistant cells, requires large amounts of DNA, and often lacks reproducibility.
The ability to deliver exogenous nucleic acids into cells allows researchers to study gene expression (inlcuding CRISPR/Cas9), RNAi gene silencing, and generate stable cell lines. Alternatively producer cells can be used for virus production, antibody/protein production, and gene therapy.
Successful delivery of nucleic acids is affected by several common factors including cell passage number, cell confluency, quality of DNA, DNA:Reagent ratio, complex formation time, and post-transfection incubation time. To learn more about optimizing, including our new our Optimization Protocol (PDF), visit our Optimization Tips from the Bench.