Stable vs. Transient Transfection of Eukaryotic Cells

Transfection, the process of introducing foreign genetic material into a eukaryotic cell, is an important tool for many cell and molecular biologists, as well as anyone studying the effects of altered gene expression on cellular physiology. Getting nucleic acids of interest—whether plasmid DNA or various types of RNA (messenger, short interfering or micro)—into a cell without killing it can be a challenge. The means of accomplishing the journey that the genetic material must take—through the plasma membrane and into the cytoplasm of target cells—varies. For example, physical methods of transfection include electroporation, microinjection and biolistic delivery with a gene gun. Chemical transfection methods may use calcium phosphate co-precipitation, diethylaminoethyl-dextran or cationic lipid-based transfection reagents.

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All of these methods address the main challenge of transfection: encouraging negatively charged nucleic acid molecules to approach and then enter negatively charged plasma membranes. Physical transfection methods avoid this electrostatic conflict by briefly pushing a hole through the membrane (or in the case of electroporation, opening holes in it) to insert the nucleic acids. Chemical methods rely on the positively charged transfection reagents forming a layer around the negatively charged nucleic acids to neutralize their charge. These events lead, hopefully, to transfection. But are you looking for transient or stable transfection?

Transient transfection

Transiently transfected cells express the foreign gene but do not integrate it into their genome. Thus the new gene will not be replicated. These cells express the transiently transfected gene for a finite period of time, usually several days, after which the foreign gene is lost through cell division or other factors.

How can you tell whether, or which, cells were transfected successfully? Usually a reporter gene is included in transfection plasmids (or along with other types of nucleic acids, if not using plasmids). Reporter genes indicate the presence of the gene of interest within the cells and can usually be assayed about one to two days after transfection.

Stable transfection

Generating stably transfected cells begins with a transient transfection, followed by an infrequent but important and serendipitous process. In a small proportion of transfected cells, the foreign gene is integrated into the cells’ genome. The hallmark of stably transfected cells is that the foreign gene becomes part of the genome and is therefore replicated. Descendants of these transfected cells, therefore, will also express the new gene, resulting in a stably transfected cell line.

When developing a stable transfection, researchers use selectable markers to distinguish transient from stable transfections. Co-expressing the marker with the gene of interest enables researchers to identify and select for cells that have the new gene integrated into their genome while also selecting against the transiently transfected cells. For example, a common selection method is to co-transfect the new gene with another gene for antibiotic resistance (such as the neomycin resistance gene, neo) and then treat the transiently transfected cells with the appropriate antibiotic for selection (such as geneticin or G418 for neo-transfected cells). Only the stably transfected cells with resistance to the antibiotic will survive in long-term cultures, allowing for the selection and expansion of the desired cells.

Which one should you use?

Deciding whether you need stable or transient transfection depends in part on whether your experiment is short- or long-term. To study gene expression using transient transfection, you can harvest the cells about 24 to 96 hours after transfection. The actual time depends on individual factors, such as the cell type and how long the expression of the reporter gene takes to level off. Thus transient transfection often is used for studying the effects of short-term expression of genes or gene products, such as gene knockdown or silencing with inhibitory RNAs, or protein production on a small scale. Transient transfection with mRNA can deliver even more rapid results than with conventional plasmid DNA, because mRNA can be expressed outside of the nucleus; in some systems, it is possible for transfected mRNA to be expressed only minutes after transfection. In contrast, stable transfection is more useful when long-term gene expression is required. Because the stable transfection of cells is a longer and more arduous process, it is reserved for research that definitively demands it, such as protein production on a large scale, longer-term pharmacology studies, gene therapy or research on the mechanisms of long-term genetic regulation.

Although the production of large amounts of recombinant proteins with proper folding and post-translational modifications (which is not available when expressing recombinant proteins in bacterial cells) traditionally has been limited to stably transfected cells, recent advances in transient transfection and cell culture methods are changing this. Ubiquitous cell culture lines such as HEK293 and CHO cells, modified to grow in suspension rather than in plated culture conditions, can now be transiently transfected to produce large amounts of recombinant proteins, as well.

As the development of stably-expressing cell lines can be laborious and challenging, the ability to use transient expression when appropriate is beneficial. Along with new transfection techniques will undoubtedly come the further refinement of both transient and stable transfection methods, enabling researchers to accomplish gene expression tasks as efficiently as possible.