Non-viral nucleic acid delivery, or transfection, forms the basis of many cell biology experiments, therefore understanding and optimizing transfection is critical to achieving meaningful results. Laboratories eager to maximize their return on scarce resources and the emergence of primary cells, stem cells, and for-purpose biomanufacturing expression systems —many of which are difficult to transfect—underscores the need for optimization from the earliest stages of experimentation.
Most transfection methods involve chemical reagents, typically cationic lipids, polymers, or liposomes that interact with or encapsulate negatively charged nucleic acids. Transfection reagents overcome electrostatic repulsions between genetic material and cell membranes to overcome the plasma membrane barrier. Many transfection reagents also incorporate functional groups that facilitate release from endosomal membranes.
Chemical reagent-based methods dominate transfection due to simplicity, reliability, reproducibility, and applicability for a wide range of cell types. Figure 1 illustrates a typical, reagent-based transfection protocol.
The process of chemical transfection as outlined in Figure 1 includes: (1) dilution of the transfection reagent and (2) exogenous nucleic acid, (3) complex formation (i.e., incubation of the reagent with nucleic acid, (4) addition of the complex to cells, and finally cell harvest and assay.
After formation, cationic transfection complexes encounter cells through electrostatic interaction with the negatively charged cell membrane. From there they enter the cell through endocytosis and are subsequently released into the cytoplasm. Delivery to the cytoplasm is sufficient for RNA (e.g. siRNA and mRNA) functionality; however, plasmid DNA requires import into the nucleus for gene expression to occur.
Figure 1. Overview of transfection process utilizing a chemical transfection reagent
Chemical composition of the transfection reagent and physical characteristics of transfection complexes are critical for successful gene transfer. Uptake is influenced by the charge ratio—the net charge carried by the transfection complex, defined as the arithmetic ratio of positively charged transfection reagent moieties to negative charges from the DNA. Optimizing charge ratio is critical when optimizing a transfection protocol and involves titrating the volume of the transfection reagent per microgram of DNA as in the example below (Figure 2). The optimal reagent-to-DNA ratio is often dependent on the cell type.
Note: Master mixes contain a 10% surplus volume for each component to reduce pipetting error and ensure that equivalent volumes can be transferred to replicate wells.
Note: It is important to include an untransfected Cells Alone Control (C1) to assess cell health following transfection. Additional controls to consider include: Reagents Alone, DNA Alone, and Medium Only.
Figure 2. Plasmid DNA transfection optimization and layout for a 12-well plate
Transfection optimization requires monitoring cells for function, including gene transcription and translation, and the ability to carry out normal cellular processes. Of concern is the balance between gene expression and transfection-induced cellular toxicity. Cell death and toxicity not only affects cell morphology but also critical cellular pathways. Since cell types respond differently to transfection, users should not assume that reagents and/or conditions that enable high efficiency transfection in one line will be effective in another.
Figure 3 illustrates differences in expression and toxicity profiles for two common cell types, A549 and MDCK cells. Luciferase activity was measured post-transfection of a luciferase coding plasmid using different chemical transfection reagents, TransIT-X2®, Lipofectamine 2000, and Lipofectamine 3000. Toxicity was measured through the release of a cytoplasmic protein, lactate dehydrogenase (LDH), into the supernatant. Different cell types have varying expression and cytotoxicity profiles that are greatly influenced by the transfection formulation.
Figure 3. Expression and cytotoxicity profiles of A549 and MDCK cells post-transfection
Fortunately, transfection optimization is not difficult. Following the workflow illustrated in Figure 1, an important factor to consider is conditions during transfection complex formation. Since serum contains nucleases and other components that can interfere with this interaction, investigators should use only serum-free media at this stage. Similarly, antibiotics (and other highly charged molecules) can disrupt the charge balance thereby inhibiting complex formation. Once nucleic acids are complexed by transfection reagents they are protected from the activity of nucleases and charged species.
Other factors to consider during optimization are: (1) nucleic acid purity (higher is better and must be endotoxin-free), (2) nucleic acid concentration (needs to be determined precisely), (3) complex formation time (typically 15-30 minutes but can also be dependent on application), (4) cell confluence (avoid too low or too high), and (5) harvest timepoint (based on application).
Cell confluence can lead to toxicity if it is too low, or conversely, if the cells are over-confluent (> 95%) the efficiency can be impaired because the cells are not actively dividing during the transfection process. Good practice is to aim for 80-90% confluency at the time of transfection. For reproducibility, transfect at similar cell confluence between experiments.
Harvest times differ depending on the experimental application. Gene knockdown and overexpression tend to require longer incubations, while mRNA translation experiments are generally ready for harvest in less than 24 hours. Virus production harvests usually occur 48-72 hours post-transfection, and large-scale protein production runs can vary from 2-14 days after transfection.
Optimization of transfection parameters is not an end, but a means of maximizing resources. It is an exercise in balancing time, cost, efficiency, and output, which includes not just obtaining the most transfected cells with the least toxicity but enabling successful downstream applications.
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DNA Transfection Optimization Protocol