How to Generate Stable Cell Lines

Transfection is a technique used to introduce foreign DNA into eukaryotic cells. Commonly, transfection is used to express a specific protein in cells, either transiently or permanently.

96 well plate containing cell cultures for DNA transfected - image by Sofiaworld

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Certain therapeutic proteins need to be processed for transfection, and this is easier to achieve using a eukaryotic cell if the protein is of eukaryotic origin. Alternatively, the protein could be tagged in some way, so that its interactions with other proteins can be studied, or the activity of certain promoters or enhancers could be studied by analyzing the level of the tagged protein produced from different promoters/enhancers.

Transfection can also be used to inhibit genes. This is achieved by the process of RNA interference, RNAi. RNAi occurs when microRNAs (miRNAs) form a RNA-Induced Silencing Complex (RISC), which attaches to the miRNA’s complementary sequence on the genome to prevent it from being expressed. miRNAs can be encoded for by vectors, which can then be added to cells to then inhibit the expression of the target gene.

How long does transfected DNA stay in a cell?

There are two ways of transfecting cells: transiently and stably. In transient transfection, the foreign DNA only lasts for a few days in the transfected cell. In stable transfection, the foreign DNA lasts for much longer, and is passed on to the next generation when the cell divides. In most cases, this is because the foreign DNA becomes incorporated into the genome, but sometimes non-genomic DNA can be maintained stably.

How do you succeed in stable transfection?

To successfully create a stably transfected cell line, it is important to consider the most effective way to deliver the foreign DNA into the cell, and how to select for cells which have gained this DNA. Around 1 in 104 cells will integrate foreign DNA when transfected, but this can be changed - it has been shown that linear DNA leads to a better efficiency of transfection, for example.

It is also beneficial to have a marker, which can be used to select the transfected cells, for example resistance to certain drugs or an essential gene that is missing from the cells being transfected. This means that, once transfected, the relevant selection method can be applied and only those that have been successfully transfected should survive. Thus, only the transfected cells should be protected from the drug, or be able to survive as they have the necessary gene to cope with the changed growth condition.

Viruses can also be used to create stable cell lines. Some vectors, which are compatible with lentivirus contain markers for selection, such as puromycin/blasticidin resistance, and thus when the vector is transduced by the lentivirus these can be used to select for successfully transduced cells.

An example of a stable transfected cell line

Certain cellular processes, such as signal transduction pathways, rely on interaction of proteins, and this could be transient. One method which can be used to study these interaction is Bioluminescence Resonance Energy Transfer (BRET) assay. Briefly, BRET assays involve the interaction of Renilla luciferase (RLuc) donor and a Green Fluorescent Protein (GFP) acceptor, and these are tagged onto various proteins to see if an interaction occurs between these proteins.

Commonly, the RLuc and the GFP are encoded onto two separate plasmids, which are then co-transfected into the cells. This typically leads to a transient transfection, and this could impact the BRET assay. The variation of the assay can be wide between repeats due to variation in transfection efficiency, and also the expression of the foreign DNA from a transient transfection may be high.

To overcome these problems, Savage and co. devised a method of creating a stably transfected cell line. To achieve this, they created a specific vector, which they called BIVISTI by using Gateway technology. When compared to transiently transfected cells, these stably transfected cells showed an increased response with less variability.

Further Reading

Last Updated: Dec 11, 2018

Dr. Maho Yokoyama

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Dr. Maho Yokoyama

Dr. Maho Yokoyama is a researcher and science writer. She was awarded her Ph.D. from the University of Bath, UK, following a thesis in the field of Microbiology, where she applied functional genomics to Staphylococcus aureus . During her doctoral studies, Maho collaborated with other academics on several papers and even published some of her own work in peer-reviewed scientific journals. She also presented her work at academic conferences around the world.

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