Scientists have access to several approaches for developing cell lines. A researcher can choose between the traditional methods that have been in use for decades and powerful new technologies for creating cell lines through genome editing. Each method has at one time or another been preferred for its particular strength or advantage; while some tried and true methods have fallen by the wayside with the adoption of newer technologies, many older protocols are still in use alongside next-generation methods.
Traditional cell line development approaches
Most cell biologists are familiar with the non-genome-editing approaches used to develop cell lines – single cell cloning, overexpressing genes or transgenes, cell line adaptation through media changes, and stem cell differentiation. Each method has its advantages and disadvantages.
Single cell cloning incubates cells individually and looks for potentially useful variations. This cloning method is straightforward but takes a long time and has limited impact. Similarly, cell line adaptation gradually changes the conditions in which the cells are grown, such as media or cell density; this method is also uncomplicated but time consuming. By contrast, gene/transgene overexpression inserts foreign DNA into cells. While this method produces quicker results, the new genetic material is difficult to control because the transcription of the exogenous gene is always on.
An alternative to cloning methods is differentiating stem cells. Versatile cell types such as mesenchymal stem cells and induced pluripotent stem cells can be differentiated to create unique cells that might otherwise be difficult to isolate and produce in large quantities. The specialized cells that result from this method can benefit toxicology and cancer researchers searching for difficult-to-acquire, physiologically relevant cell models.
What choices do researchers have for gene editing?
Gene editing is a relatively recent advance that has been capitalized on by scientists to create unique cell lines. Overall, the advantage of these technologies is that targeting is specific. However, the downside is that a certain amount of technical expertise is required to make the method work. In short, an endonuclease is required to create a double-stranded DNA breaks in your gene of interest. When the cell tries to repair the damage, the sequence can be changed via the addition of exogenous DNA or RNA containing desired sequence. This process can add any cell property with a specific genotype/phenotype connection. If researchers know how a gene controls a function, they can change that function. This is relatively easy to accomplish when a gene controls one function, but much more challenging when hundreds of genes work in concert.
Early approaches to gene editing, including zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), are effective but cumbersome. Researchers must develop a new series of proteins for each edit. Since proteins are difficult to design and test, each edit can be expensive, and researchers must make choices, making these approaches less efficient than CRISPR/Cas9 genome-editing technology.
CRISPR technology employs a nuclease protein (Cas9 or similar) paired with an interchangeable guide RNA molecule to locate and cut a gene. Short RNA molecules are cheap, easy to make, and can be made to target any gene of interest in any organism or cell line. The combination of a powerful universal nuclease protein with a simple, interchangeable, and highly specific guide RNA makes CRISPR the most cost-efficient method of gene editing to date. Because the guide RNAs can be exchanged so readily, researchers can screen large sets of targeting molecules for maximum gene editing efficiency. Furthermore, the lower costs and increased editing efficiency of CRISPR gene editing means that more complex modifications like introduced point mutations, targeted deletions, or gene fusions can be engineered significantly increasing project costs.
While CRISPR-Cas9 gene editing is superior in many ways, the current patent and licensing landscape remain unsettled and many companies are still using ZFN and TALEN.
How many changes can you make?
Researchers can edit as many sites as time and resources allow. If the editing efficiency is low or they are performing a more complex edit, scientists should edit one gene at a time, isolate the successfully edited cells and then make another edit. If editing efficiency is high and the edits are more straightforward, researchers can perform several simultaneously and identify, isolate, and expand the cells with the intended edits.
Engineer an edit with a known effect
There is always a chance an edit will damage the cell. Even well-understood edits can have negative effects, especially when targeting multiple genes with unpredictable interactions.
To avoid these consequences, it’s best to engineer an edit with a known effect. These edits generally mimic a phenotype in nature, such as a cancer mutation, natural gene variant or disease mutation. These edits can also mirror a specific but nonlethal effect that’s been seen in a cell line or animal model.
If the gene of interest is not well-studied, researchers can make educated guesses on which edits will generate the desired effect. Another approach is editing several targets and screening for the desired outcome. Unfortunately, this can consume resources without producing anything useful.
Why haven’t companies used editing to develop an ideal cellular expression system?
Each protein product is different, and there are many parameters to consider – from the desired characteristic to the final product’s ultimate application. Edits that help a cell produce therapeutic antibodies don’t necessarily help them produce viral proteins for vaccines.
Since the relationship between genes and protein production capacity is complicated, there’s generally no single change, or series of changes, that will greatly improve a specific protein’s expression.
In addition, much work has been done on protein expression systems using clonal selection, cell line adaptation and older gene editing methods. Because so many legacy methods exist, and scientists are reluctant to replace a working technology, it’s unlikely that even an ideal system could displace current practice.
While large biotech and pharma companies have the resources to overcome these barriers, they are reluctant to create an ideal system because cells (the product) are replicable and could be distributed without licensing.
Elizabeth Turner Gillies PhD, Scientist, ATCC