Transfection Methods for Cell Culture

The choice between transient or stable transfection will usually depend on your goals, cell type and the facilities available to you. In short, transient transfection exerts a temporary influence on your cells, whereas stable transfection leads to permanent genetic changes that are usually passed on to future cell progeny. More details on both transient and stable transfection are provided below.

Transient Transfection

For most transfection applications, a transient influence on gene expression or cell function is necessary, sufficient or preferred. Examples of such applications include short-term gene expression studies, gene knockdown studies and small-scale protein production. To achieve transient transfection, you must use factors that will not be incorporated in the host cell’s genome. This can include using DNA, proteins, mRNA or non-coding RNAs, all of which will degrade over time or will be diluted out through cell division. As such, transient transfection only influences your cells over a short time window spanning a few hours to a few days. 

Stable Transfection

To achieve stable transfection, you must use genetic constructs that will be incorporated into the genome of the host cells (either into the chromosomes themselves, or as extra-chromosomal episomes), so that they will pass these new genes onto their offspring. The constructs must also carry selection markers, so that you can identify which cells have been successfully transfected. Stable transfection is often required for large-scale protein production, research into long-term gene regulation, the generation of stable cell lines, and for gene therapy. As the method requires successful DNA integration into the host genome, it is often much harder to achieve than transient transfection, and typically has lower transfection efficiency. Stable integration can occur randomly with plasmids, actively at random sites with help of transposases or viruses, or site-specifically when using genome editing tools like CRISPR.

You can use DNA, RNA or proteins to transfect your cells, and each has its own advantages and disadvantages. Additionally, certain cell types or applications may only work with a specific transfection substrate, and you will often need to reoptimize your transfection conditions when switching substrates. More details on each transfection substrate are provided below.
 

Plasmid and Linear DNA Transfection  

Plasmid DNA is commonly used for transfection (although linear DNA can also be used). In both cases, you should ensure that your DNA is as pure as possible, with any contaminating lipids, salts, proteins, nucleotides or other factors removed via DNA purification. The best way to obtain high quality DNA is by using a purification method based on anion exchange technology which includes an additional endotoxin removal step. This helps to ensure optimum transfection efficiency and cell viability. When transfecting DNA purified with silica membranes, there is a high risk, on poor transfection efficiency and cell viability, further downstream results might not be trustable. 

DNA is the substrate of choice for a wide range of applications, as it is relatively stable, easy to handle and cost effective to produce. DNA vectors are also the best option if you are looking to achieve stable transfection via incorporating your construct into the target cell’s genome. However, as DNA constructs must make their way into the nucleus to induce gene expression, they are slower to drive protein production than mRNA (which exerts its effects as soon as it enters the cytoplasm, see below). In all cases the toxicity of the expressed protein has an impact on the transfection result, no matter if DNA, mRNA or the protein itself was transfected. When transfecting plasmid DNA, also the plasmid backbone and the promoter can have an influence on the transfection result. 

RNA Transfection

The RNA used for transfection can take several forms, depending on the application. For example, mRNA is an effective substrate for inducing rapid protein expression, as it enables results to be achieved more quickly than using DNA. It is also possible to transfect cells with regulatory or non-coding RNAs to modify the expression of endogenous genes (e.g. gene knockdown via RNA interference). 

In general, RNA transfection has a higher efficiency than DNA transfection as it just needs to enter the cytoplasm. Furthermore, with DNA-sensitive cells, cell viability is often much better when using RNA. As already said before, the toxicity of the encoded protein has an impact on the transfection result as well. However, protein production may be lower overall, as mRNA is more unstable than DNA and no new RNA is produced by the cell following transfection. The general instability of RNA can also make it more challenging to work with. Meanwhile, production can also be more cumbersome (as the mRNA must be transcribed from a DNA template, introducing an extra step) and/or expensive (if the RNA will be chemically synthesized). 

Some applications are not well-suited to RNA transfection. For example, to create induced pluripotent stem cells (iPSCs) using mRNA, you must deliver it repeatedly over time. In addition, RNA cannot be used for stable transfection, as it will not be integrated into the host cell’s genome. 

Protein Transfection 

Protein transfection is used for specific applications, such as the introduction of Cas9 enzymes for gene editing via CRISPR, and the use of transcription factors to reprogram cells to generate iPSCs. The advantage of using proteins is that they usually exert their influence on the cell immediately and the quantity can be controlled much better. However, proteins vary greatly in terms of size, shape and charge, so transfection conditions must often be specifically optimized on a per-protein basis. Another practical consideration is that proteins must either be made and purified or purchased from a supplier – both options can be costly, especially when compared to generating DNA or RNA substrates.

There are different transfection methods existing, all having their advantages and disadvantages. Below, we shortly describe the different transfection methods ranging from viral transfection, to chemical transfection methods like calcium-phosphate precipitation, lipofection and DEAE-dextran, as well as electroporation. We end this paragraph with our improved electroporation technology, which is also called Nucleofection.

Viral Transfection 

Viral transfection (also called viral transduction) takes advantage of the natural ability of certain viruses to carry foreign genes into host cells. Depending on the virus used (adenovirus etc.), you can achieve either stable or transient transfection. Using viral methods for primary cell transfection and cell line transfection generally yields high efficiencies.  But there are several challenges to overcome when using viral transfection. For instance, it is often a time-consuming process that raises biosafety considerations (depending on the type of virus being used). In addition, the size of your DNA or RNA insert is limited to about 10 kb for most viral vectors. This is small when compared to non-viral vectors, which can carry up to 100 kb. A range of viral vectors can be used for transfection, each of which has different advantages, disadvantages and applications.

If you’d like to learn more about the different viruses used, download our free whitepaper: ‘An Introduction to Transfection Methods – Technical Reference Guide’.

Calcium Phosphate Transfection

Transfection requires the delivery of material through the cell membrane, which is negatively charged. As nucleic acids such as DNA and RNA are also negatively charged, they repel each other, inhibiting their uptake by the cell. One way to overcome this challenge is to use positively-charged carrier molecules to ferry negatively charged substrates close enough to the cell membrane to be internalized via endocytosis. Calcium phosphate Transfection is one of the chemical transfection methods using this principle. 

This technique involves mixing your DNA construct with calcium chloride in a buffered saline/phosphate solution. When the mixture is incubated at room temperature, DNA-calcium phosphate coprecipitates form. As these coprecipitates can adhere to the plasma membrane, they are thought facilitate endocytosis. Calcium phosphate transfection is widely used for both transient and stable transfection applications across a range of cell types, especially as the required reagents are affordable and accessible for most labs. 

Unfortunately, this method can be toxic to cells (especially primary cells) and the transfection efficiency is relatively poor compared to most other methods. In addition, the reaction used to generate the coprecipitates is sensitive to slight changes in pH, temperature and buffer-salt concentrations, leading to unreliable results. 

DEAE-Dextran Transfection

It is also possible to use diethylaminoethyl-dextran (DEAE-dextran) to facilitate transfection. DEAE-dextran is a polycationic derivative of dextran (a carbohydrate polymer). When mixed with the DNA, the resulting complex is carried into contact with the negatively-charged plasma membrane by the excessive positive charge provided by the polymer. As for calcium phosphate, this close proximity to the membrane is thought to facilitate endocytosis into the cell. While using DEAE-dextran is relatively simple and low cost, the transfection efficiency of the method is generally low for many cell types. It can also be cytotoxic and is not usually suitable for generating stable cell lines.

Lipofection

Lipofection is the most commonly-used chemical method of transfection. The approach involves combining cationic lipids with other molecules to create unilamellar liposome vesicles that carry a positive charge. The exact molecular mixture has varied over time, as lipofection methods have improved. 

Regardless of the makeup of the vesicles, lipofection is designed to package negatively charge molecules like nucleic acids in a positively-charged vesicle, so that they can get closer to the cell membrane (where they are presumably taken up by endocytosis). As such, you must mix your construct or molecule of interest with the vesicle mixtures ahead of transfecting your cells. 

The approach can be successfully used to transfect a wide range of cell types at relatively low cost (although cost and reaction conditions can increase as other polymers and antibody-conjugates are added). Furthermore, lipofection can transfect cells using DNA of all sizes to produce both stable and transient transfection, as well as deliver RNA and proteins into cells. The main drawback of the technique is the low transfection efficiency achieved when it is used for primary cell transfection, stem cell transfection and when working with suspension cell lines (mainly due to the fact that it can be cytotoxic and relies on cell division for success). 

If you’d like to learn more about Lipofection and other chemical-based transfection methods, download our ‘Transfection Methods – Technical Reference Guide’ here.

Electroporation

Electroporation is a physical method of transfection that involves first suspending your cells and DNA construct in an electroporation buffer. High-voltage pulses of electricity are then applied to the mixture, which creates a potential difference across the cell membrane. This introduces temporary pores that allow the exogenous DNA to enter the cell. To successfully perform this technique, you usually need to test and optimize the duration and strength of the pulses against your specific cell type and construct. 

As for all transfection methods, electroporation has its advantages and disadvantages. On the plus side, you can use it to transfect large DNA fragments and achieve good transfection efficiencies using cell lines. However, transfection efficiency in primary cells is low, mostly due to the high mortality rates caused by the electric pulses. Furthermore, relatively high substrate amounts are required to achieve efficient transfection.

The Nucleofector^TM Technology is based on two main components. The first is the Nucleofector^TM Device, which offers a unique portfolio of electrical parameters, displayed as distinct programs that are adapted to the requirements of specific cell types. This means that the user does not need to optimize the pulse parameters manually (saving a lot of time, cells and reagents). The second component is provided via a range of Nucleofector^TM Kits, each of which contains cell type-specific Nucleofector^TM Solutions, Cuvettes and Pipettes. 

How can you choose the best transfection method for your cells? Well, the ideal approach should offer high transfection efficiency, low toxicity and yield reproducible results. From a practical perspective, it should also be as quick and straightforward as possible to carry out.

To help you select the optimal transfection tool, consider these key questions:

• Which cell type are you using – primary cells, stem cells, cell lines, adherent cells, suspension cells etc.? 
• Do you need to achieve stable or transient transfection?
• What molecule will you transfect (DNA, RNA or protein)?  
• What is the transgene packaging capacity (i.e. what is the size of your construct)?  
• Are there any general safety concerns? If so, what are they?  
• Have you considered all the practical issues (e.g. cost, time, facilities/expertise available etc.)?

To further help you select the best way to transfect your cells, we’ve created a handy table comparing all the available transfection methods, highlighting their relative advantages and disadvantages (see below). You can find more information on transfection techniques by downloading our free pdf resource: ‘An Introduction to Transfection Methods – Technical Reference Guide’.