Transfection

Transfection

Summary

Transfection is the process of introducing new nucleic acids into a eukaryotic cell, similar to transformation in bacteria. The most common purpose of transfecting eukaryotic cells is to alter gene expression so that a protein of interest will be expressed within the cell. The nucleic acids being introduced into the cell usually inserted in the form of a plasmid vector or mRNA. There are many methods used for transfection, including electroporation, the use of nanoparticles to undergo transfection and cationic lipid transfection (liposome based transfection).

In our Lab

Currently in our lab, we use liposome based transfection to introduce CRISPR DNA (called CRIPSR method on thermo fisher) to introduce GFP + Cas9 into our cultured melanoma cells. Our transfection reagent is lipofectamine, which forms a cationic liposome that surrounds and forms a complex with the plasmid DNA to allow it to traverse the lipid bilayer of the cell. Specifically, the lipofectamine (liposomes) will form a positively charged lipoplex around the negatively charged DNA. The positively charged exterior of the lipoplex will allow it to fuse with the negatively charged lipid bilayer in the cell membrane, thus delivering the contents of the lipoplex into the cell (the DNA of interest). The of use of transfections to introduce DNA into eukaryotic cells is important because eukaryotic cells do not have their own mechanism of up taking nucleic acids from their environment as some prokaryotic cells do.

In order for gene expression to occur, the transfected DNA must reach the nucleus. However, in many cells, the transfected DNA will never reach the nucleus, thus explaining the ~2% success rate of transfection when observing cells transfected with eGFP under fluroescence. An example of how the DNA can reach the nucleus is by being trapped inside the newly formed nuclear envelope in dividing cells. Reasons the DNA will not reach the nucleus are usually due to errors in the delivery process within the cell.