What is Transfection?

Transfection is the process of delivering nucleic acids and small proteins into eukaryotic cells. This can be achieved by using chemical transfection reagents, electroporation (gene electrotransfer), viral transduction, and several other less common methods. The ultimate goal of transfection is to deliver nucleic acids into cells so as to investigate gene function. This can be accomplished 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. 

Figure: Chemical Transfection of Eukaryotic Cells - 1) Transfection reagent is combined with nucleic acid to form positively charged transfection 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) Transfection 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.  

Chemical Transfection

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. 

Why is transfection important?

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.  

The Basics of Successful Transfections

Successful deliver 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, visit our DNA Transfection Optimization Tips from the Bench.