Transient transfection is a very effective way to express genes in host cells. Due to its rapid production rates, it is often used in studies with a focus on short-term expression of genes and gene products, such as in recombinant antibody production.
However, only through the use of specific cell lines, e.g. CHO cells, it became possible to accomplish high production yields of protein through transient transfection. This allows for optimized production processes and saves time and resources. The CHO cells antibody production was a major part in this.
In this article, we will provide an overview of transient transfection and how it is used. Furthermore, we will explain the differences between transient transfection and the second transfection method – stable transfection –, as well as their different uses.
As HEK293 and CHO cell lines have changed the way transient transfection serves the production of biotherapeutics, we will give you an insight of why this was possible, too.
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The results transient transfection has brought to the studies of gene products and functions in eukaryotic cells have impacted research in this sector by a substantial amount. Hence, transient transfection plays a big part in biotechnology and the development of many important biotherapeutics.
The technique continues to prove very valuable in the production of antibodies and recombinant protein through recent optimization of the process leading to higher production yields and the prevention of cell death.
Read more: What is transient transfection?
The main purpose of transient transfection is to study the effects of short-term expression of genes and gene products. As the nucleic acid is not integrated into the genome, the genetic material only exists for a limited amount of time and is not passed on.
By using mRNA instead of plasmid DNA, which is translocated into the nucleus for transcription, it is possible to achieve rapid results. Sometimes, expression shows only minutes after transfection! Through the introduction of siRNA into the genome, it is possible to conduct gene silencing experiments in a short amount of time.
However, while one of the main flaws of transient transfection was its limitation to short-term expression, recent modifications of specific host cell lines like HEK293 and CHO cells have made it possible to produce large amounts of recombinant proteins with post-translational modifications.
There are different methods to introduce an expression vector genome into a cell. In the beginning, a cell type is usually cultured in vitro. As their main purpose is to produce high quantities of proteins, it is not necessary that the cell resembles the in vivo conditions.
Afterwards, the gene of interest, often accompanied by a reporter gene, is introduced through the cell membrane without damaging the cell to achieve a synthesis.
Most commonly, the biological method is used, where transfected nucleic acids are delivered to the cell by viral vectors. However, this can also be achieved by chemical transfection methods.
In cationic lipid transfection, chemicals are added to enhance the positive charge of DNA polymer in order to penetrate the negatively charged membrane and allow for gene transfer. Physical transfection includes microinjection, optical transfection, biolistic transfection and electroporation.
The most commonly used method in transient transfection is PEI-mediated transfection, where plasmid and oligonucleotides are used.1 The different steps are as follows:
The benefits of transiently transfected cells especially come into play in rapid protein expression. It is possible to go from cloning a gene to large amounts of protein in just a few days.
As the nucleic acid is not integrated into the genome and the reaction is not taking place in the cytoplasm, transiently transfected cells are characterized by a high efficiency for a short period of time. This characterization of transiently transfected cells allows for quick results in the study of different materials, like receptor proteins for instance.
The proteins produced through transient transfection are fully post-translationally modified and active mammalian proteins. The secreted proteins can be easily purified from serum-free cell culture medium, with methods like affinity chromatography and protein polishing techniques like size exclusion or ion exchange chromatography as offered by evitria.
Read more: Benefits of transient transfection
There are two types of transfections – transient and stable transfection. While stable transfection tends to be more laborious and requires more effort and resources, it supports gene expression over a long period of time through the integration of the nucleic acid into the host genome.
This leads to permanent genetic changes that will be passed on to the next generation of cells. While using linear DNA in stable transfection causes lower DNA uptake in comparison with supercoiled DNA, it also leads to optimal integration of the nucleic acid into the host genome.
Transient transfection does not require the integration of the foreign DNA into the host cell genome. It is less complicated to perform but, at the same time, the transient transfected genome will only be expressed temporarily. While stable transfection relies on the use of DNA transfection, transient transfection can be performed using both DNA and RNA. It is most efficient using supercoiled DNA.
Transient transfection is mostly used to study the effects of short-term expression of genes, while stable transfection, due to its long-term effects, is used for protein production on a larger scale. However, with modifications on HEK293 and CHO cell lines, it has become possible to achieve larger production yields using transient transfection.
HEK or CHO cells are two of the most commonly used cell lines in mammalian cell transfection. In order to produce a large amount of recombinant protein with post-translational modifications, this was usually achieved through stably transfected cells.
However, this started to change with the use of transient transfected HEK293 and CHO cells adapted to suspension culture. Now it is possible to obtain high amounts of recombinant protein without the difficulties, resources and time involved in the process of stable cell line development.
The use of HEK293 cells as a transiently transfected expression system has proved itself valuable in multiple experiments. Through their ability to generate human-like glycosylation profiles, they can successfully be used in recombinant protein production and antibody production.
High cell division and replication rates and the fact that they can easily be manipulated qualify them as one of the main work horses in biotherapeutic research.
However, due to their human origin, they are at a higher risk of being contaminated with human-specific viruses. This can lead to immunogenicity or allergic reactions to the final product.
The many benefits of using CHO cells (Chinese Hamster Ovary cells) in transient transfection qualify them as one of the most popular cell culture lines in bio research and production among HEK293, COS and BHK cells.2 This especially applies due to their transfection efficiency.
They are easy to cultivate on large scales and show excellent growth rates as adherent culture and in suspension. Moreover, it is possible to produce high amounts of recombinant protein by using CHO cells. Through genetic optimization of CHO cells, their productivity was enhanced and toxicity risks could be decreased. Their hamster origin makes them less susceptible to human viruses.
In conclusion, it can be said that the future of transient cell transfection is full of potential. Especially the use of CHO cells opened up completely new possibilities in regards to production time and yields, which makes them the host cell line of choice for recombinant monoclonal antibody production at evitria.
And thanks to continuous optimization and new techniques, CHO cells will continue to help us make the production process of biotherapeutics more efficient. Get in contact with us for your recombinant antibody production service.