July 1, 2015 (Vol. 35, No. 13)
Andrea Toell Ph.D. Product Manager Lonza Walkersville
Improving Transfection Efficiency Is an Important Step for Enabling Effective Genome Editing
Genome editing technologies have advanced significantly in recent years, offering researchers the opportunity to precisely manipulate the genome at specific locations in a faster and more effective manner than ever before. Three genome editing technologies are commonly used at the current time: Zinc-Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas9) system. ZFNs and TALENs are engineered constructs created by fusing endonucleases to a DNA-binding domain. CRISPR-Cas9 is a system that utilizes synthetic guide RNAs to facilitate targeted DNA breaks via the action of the bacterial Cas9 endonuclease.
Each process allows researchers to target a specific locus of interest to mutate, exchange, or inactivate a gene. Using the systems, it is also possible to introduce complex synthetic gene variants or insert a completely foreign gene directly into the genome of the host cell. Importantly, any changes are made at a very specific genomic location, directly under the control of the researcher.
Genome editing is a powerful tool, with a number of exciting possible applications. However, the process inherently relies upon the transfection of the necessary components into the cells of interest in order for them to exert their effects. As a result, cell types that have traditionally proven challenging to transfect have also resisted efforts by researchers to carry out genome editing applications. This has hindered studies in a number of interesting research areas, as well as the development of targeted gene therapeutics to treat a range of diseases, including blood-borne conditions such as HIV and leukemia.
Technologies that improve the transfection of genome editing substrates into cells would both enhance studies using common cell types, as well as open up exciting new avenues of exploration in cells that have so far proven difficult or even impossible to work with.
Common transfection methodologies include lipofection, electroporation, and viral transduction. Lipofection encloses the substrate in a lipid-vesicle, which then enters the cell via endosomal uptake. Electroporation alters the permeability of the cell membrane, allowing the substrate to cross the membrane and enter the cell. Viral transduction involves encapsulating the substrate in a viral protein coat, which can then be used to “infect” the target cell and deliver the substrate.
The transfection process can be significantly optimized by utilizing an efficient transfection technology and selecting the optimal substrate, based on the target cell type and approach. It is also desirable to implement a flexible process, so that new cells and substrates can be rapidly and easily studied in the future without the need for further optimization and resource investment.
Improving Transfection Efficiency
One especially effective nonviral transfection approach is the use of Lonza’s Nucleofector™ Technology. This is an enhanced method of electroporation that enables the delivery of substrates directly into the nucleus, independently of cell proliferation. Consequently, this results in higher transfection efficiency in a wide range of cell types, including those that are hard to transfect (e.g., primary cells, stem cells and a number of specific cell lines).
To illustrate this, primary blood cells and cell lines, which are commonly used in research, were transfected with pmaxGFP™ Vector using Nucleofection and analyzed for transfection efficiency after 24 hours using flow cytometry (Figure 1, blue bars). The results show that many of these cells achieved transfection efficiencies of more than 50% using the technique, with some reaching levels as high as 90%.
In the same experiment, the cells were assessed for viability 24 hours post-transfection using a ViaLight™ Assay (Figure 1, red bars). Most cells showed high levels of viability post-transfection. The results imply that the Nucleofector Technology could offer an effective means of transfecting challenging cell types, such as those of the blood cell lineage, with genome editing DNA constructs.
Selecting a Substrate
When selecting a transfection method, researchers also need to consider the type of substrate they are using and the unique requirements of their target cell type. In many cases a DNA plasmid is used to carry genome editing tools. However, in some cases, it may be advantageous to transfect an RNA or protein substrate, rather than DNA. Examples include when working with DNA-sensitive cells, such as human dendritic cells, J774A.1 cells, or NB-4 cells, or when a limited presence of the editing tool is required.
To test the effectiveness of Nucleofection when using RNA substrates, a comparative analysis was conducted to assess the viability of DNA-sensitive cells when using pmaxGFP Plasmid DNA, GFP mRNA, and fluorescently labelled siRNA as transfection substrates (Figure 2). Cells were analyzed 24 hours post-transfection for transfection efficiency via flow cytometry and viability using the ViaLight Assay, except in the case of the siRNAs, in which the cells were analyzed 3 hours after transfection.
In each case, the viability of the cells was improved when switching to a RNA-based substrate. The data suggest that DNA-sensitive cells such as J774A.1, NB-4, and primary human dendritic cells might be more amenable to genome editing experiments if RNA is used as the substrate instead of DNA. In addition, the results show that high efficiencies are achievable when transfecting RNA or oligonucleotide substrates using Nucleofection, under the same conditions as with DNA plasmids.
Cell Type Flexibility
Selecting a transfection method that is compatible with a wide range of cells could save repeated protocol optimization and allows researchers to quickly adapt their protocols for use with additional cell types.
As demonstrated previously, Nucleofection is compatible with a wide variety of cells, including those derived from blood. The method has already been successfully used with several genome editing systems by a number of researchers (Table).
Genome editing is a powerful tool for basic research and therapeutic development. Transfection is an essential part of this process and an optimized transfection protocol is a vital prerequisite for success. Lonza’s Nucleofector Technology enables the efficient transfection of a wide range of cell types and can be used to deliver DNA or RNA substrates (and even proteins when required—data are not shown here, but see the Table for relevant studies).
Nucleofector Technology has already been used to enable success in a number of peer-reviewed studies and offers researchers a powerful tool across a number of exciting research applications.