With any transfection procedure, a critical first step is to optimize the transfection conditions. Every cell type and transfection procedure has a characteristic set of requirements for optimal introduction of foreign DNA, and these conditions have a large degree of variability even among cell types that are very similar to one another.
The single most important factor in optimizing transfection efficiency is selecting the proper transfection protocol for the cell type. Once the appropriate transfection method is selected, a transient reporter assay system can be used to optimize the procedure by transfecting a reporter gene under a variety of conditions, and monitoring the transfection efficiency by assaying for the reporter gene product.
This section provides general guidelines for optimizing calcium phosphate–mediated gene transfer, electroporation using the Neon® Transfection System, and cationic lipid mediated transfection.
Considerations for calcium phosphate co-precipitation
The primary factors that influence the efficiency of calcium phosphate transfection are the amount of DNA in the calcium-phosphate–DNA co-precipitate, the length of time the cell is incubated with the co-precipitate, and the use and duration of glycerol or DMSO shock.
Total DNA amount used in calcium phosphate transfection is usually 10–50 μg in 450 μL sterile water and 50 μL of 2.5 M CaCl2 per 10-cm dish, but varies widely among plasmid preparations as well as with different cells and media. While with some cell lines 10–15 μg of DNA added to a 10-cm dish results in excessive cell death and very little uptake of DNA, other cell lines, especially primary cells, much higher concentrations of DNA is required. Each new plasmid preparation and each new cell line being transfected should be tested for optimum DNA concentration.
The optimal length of time that the cells are incubated with co-precipitate also varies with cell type. Some hardy cell types, such as HeLa, NIH 3T3, and BALB/c 3T3, are efficiently transfected by leaving the co-precipitate on for up to 16 hours, which might kill some more sensitive cells.
A pilot experiment varying the amount of DNA, incubation time, and exposure to glycerol or DMSO shock will indicate whether the cell type is tolerant to long exposure to a calcium phosphate precipitate and whether glycerol shock should be used. Once the results of the pilot experiment are obtained, further optimization can be performed by adjusting the experimental variables even finer. For instance, if shocking the cells with 10% glycerol for 3 minutes as shown in the example below enhances transfection efficiency, an experiment varying the time of glycerol shock or using 10–20% DMSO shock might also be tried.
Table 1: Pilot experiment example for the optimization of transfection by calcium phosphate co-precipitation
|Dish (10-cm)||Reporter plasmid (μg)||Incubation (hr)||Glycerol shock (min)|
Considerations for cationic lipid-mediated delivery
Four primary parameters affect the success of DNA transfection by cationic liposomes: the amount of DNA, the ratio of transfection reagent to DNA, incubation time of the lipid-DNA complex, and the cell density at the time of complex addition. These factors should be systematically examined for every cell type and vector combination, and once optimized, kept constant in all future experiments to help ensure reproducible results.
For best results, follow the optimization protocols provided by the manufacturers of the reagent.
The optimal amount of DNA varies depending on the characteristics of the transfected plasmid (e.g., promoter, size of plasmid, origin of replication), number of cells to be transfected, size of the culture dish, and the target cell line used. In many of the cell types tested, relatively small amounts of DNA are effectively taken up and expressed. In fact, higher levels of DNA can be inhibitory in some cell types with certain cationic lipid preparations. In addition, cytotoxicity may result if a plasmid encoding a toxic protein or too much plasmid with a high expression rate is used.
The overall charge of the transfection complexes is determined by the ratio of transfection reagent to DNA. The negative charge contributed by phosphates within the DNA backbone needs to be offset by the positive charge contributed from the transfection reagent for both good complex formation and for neutralizing the electrostatic repulsion imparted on the DNA by the negatively charged cell membrane.
The optimal ratio of transfection reagent to DNA is highly cell type-dependent. As a starting point, the amount of transfection reagent should be varied while keeping a constant plasmid DNA concentration (for example, 1:1, 3:1 and 5:1 ratios of volume to mass). Additional benefits may be derived by maintaining the ratio and increasing the amount of plasmid added.
The optimal incubation period of cells with the transfection complexes depends on the cell line and transfection reagent used. In general, transfection efficiency increases with time of exposure to the lipid reagent-DNA complex, although toxic conditions can develop with prolonged exposure to certain lipid reagents, requiring removal by centrifugation or dilution with fresh medium after a given incubation period to minimize cytotoxic effects. However, newer and gentler transfection reagents such as the Lipofectamine® 3000 reagent do not necessitate complex removal or dilution after transfection.
When using cationic lipid reagents that require adding or replacing the medium, vary the incubation time after complex addition (e.g., 30 minutes to 4 hours, or even overnight) and monitor cell morphology during the this interval, particularly if the cells are maintained in serum-free medium as some cell lines lose viability under these conditions.
Cell density also affects overall transfection efficiency. To achieve transcription and ultimately protein production, nuclear deposition of DNA is required, which is largely dependent on membrane dissolution and reformation during mitosis, requiring that the cells have to be actively dividing.
For adherent cells, the best efficiency is often attained at a confluency of 80%, but protocol recommendations may range from 40–90%. For suspension cells, we recommend splitting the cells the day prior to transfection to ensure that the cells will be in optimal physiological condition for the transfection procedure. The optimal density is highly dependent on cell type and reagent-specific toxicity, and should be
Figure 1: Example transfection workflow using the Lipofectamine® 3000 transfection reagent.
Considerations for electroporation
Electroporation is mainly dependent on the combination of three electric parameters: the pulse voltage, pulse width, and pulse number. Perhaps because it is not a chemically based protocol, electroporation is less affected by DNA concentration; however, it requires almost five-fold more cells and DNA compared to calcium phosphate-mediated transfection. Generally, 1–5 μg of DNA per 107 cells is sufficient, and there is a good linear correlation between the amount of DNA present and the amount taken up.
The objective in optimizing electroporation parameters is to find a pulse that maintains 40–80% survival of the cells. The pulse width is determined by the capacitance of the power source and the extent to which this can be varied depends on the electronics of the power supply generating the pulse. If excessive cell death occurs, the length of the pulse can be lowered by lowering the capacitance.
Keeping cells on ice often improves cell viability and results in higher effective transfection frequency, especially at high power which can lead to heating (Potter et al., 1984). However, some cell lines electroporate with higher efficiency at room temperature under low voltage/high capacitance conditions (Chu et al., 1991).
The Neon® Transfection System, available from Life Technologies™, is pre-programmed with 18- and 24-well optimization protocols that allow quick optimization of electric parameters for many adherent and suspension cell lines within days. Cell line-specific optimized protocols for the Neon® Transfection System can also be conveniently downloaded to maximize transfection efficiencies for many commonly used cell types.