The Who, the Why, and the of How of transfection?
Who: Scientists rely on transfection as a powerful technique to modulate gene expression in eukaryotic cells in vitro and in vivo.
Why: transfection is the process that allows exogenous nucleic acids to bypass the cell membrane to enter into cells. Exogenous nucleic acids commonly used are plasmid DNA, RNA, siRNA and oligonucleotides. Once delivered into cells, nucleic acids modulate gene expression by driving overexpression or silencing of a gene of interest.
Gene overexpression is an indispensable tool for several applications, from understanding the role of gene of interest (gene studies, high-throughput screening), to the production of biologics such as antibodies (protein production) and recombinant viral particles, particularly for therapeutic purposes (virus production for gene & cell therapy).
Gene silencing is a method used to prevent expression of a gene of interest. The expression of a gene can be partially reduced (gene knockdown) or completely blocked (gene knockout). Because any gene can potentially be targeted, gene silencing is a prevalent technique used to develop gene-based therapies to address monogenic pathologies, cancer and in immunotherapy strategies.
How: Transfection of nucleic acids into cells cannot be achieved without the help of a transfection method. There are several physical methods that exist such as electroporation, sonoporation or microinjection but these processes are complex and relatively toxic for mammalian cells. To solve these issues, chemical-mediated transfection offers a great alternative: easiness of use, high transfection efficiency and excellent cell viability.
Chemical-mediated transfection
1) encapsulation of genetic material with transfection reagent
Nucleic acids are negatively charged due to their polyphosphate backbone and are thus able to interact with positively charged transfection reagents (polymers or lipids). This results in the formation of transfection complexes or nanoparticles, which protect nucleic acids from nuclease-mediated degradation.
2) Cellular uptake of nanoparticles
Most cells express negatively charged heparan sulfate proteoglycans on the external surface of their cell membrane, with which positively charged transfection complexes are able to interact. This interaction is key to trigger cellular uptake via an endocytosis process.
3) Release into the cytosol and if needed transport into the nucleus for transcription
Upon cellular uptake, transfection complexes are sequestrated into intracellular vesicles. Our transfection reagents are able to induce the release of the nucleic acids into the cytoplasm through vesicle membrane rupture or fusion. Most nucleic acids (oligonucleotides, siRNA, mRNA, etc…) stay in the cytoplasm where they are active. In case of gene transfer, plasmid DNA is transported into the nucleus for transient expression) which can become permanent after genome integration (stable expression).
Troubleshoot your transfection
When getting started with transfection, having additional tips and advice can be a real advantage to progressing seamlessly with your experiments. Even if you are not a novice in transfection, changing plasmid DNA construct, working with a new cell line or isolated primary cells can require fine-tuning. As transfection experts, we are always available to help you. Simply reach out to our Scientific Support Team:
In the meantime, you can also search through our regularly updated:
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FAQ to answer the frequently asked questions we have received regarding our reagents and transfection experiments.
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Cell Transfection Database with over 6000 publications, 1000 cell lines and primary cells. Transfection protcocols are available.
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