by Catherine Shaffer
Transfection is still a major bottleneck for many researchers. Primary cells are valued because they more closely model live tissue, but they are difficult to transfect. Successfully completing the challenge of transferring plasmid DNA or RNA into a cell requires careful selection of transfection reagent as well as attention to cytotoxicity and transfection efficiency. An inefficient transfection or high toxicity to the cells can torpedo the whole project. Other common challenges in transfection include degradation of DNA by nucleases and effective transport of DNA into the nucleus. These are all barriers that an efficient transfection reagent can help to overcome.
There is no one-size-fits-all solution for transfection that works for all nucleic acids, all applications and all cells with equal efficiency. Manufacturers of DNA transfection products strive to identify and test different reagents for specific applications.
How can efficiency be optimized?
Highly efficient nucleic acid transfer is the goal of every transfection, but efficiency must be balanced against cytotoxicity. There's a fine line between harmlessly inserting DNA and poisoning your cell. Transfection is a traumatic event for the cell.
The ultimate goal is to get high transgene expression while maintaining the health of the cell. An ideal transfection reagent delivers high efficiency, low cytotoxicity and high reproducibility. Different cell types have different transfection properties, and established cell lines are easier to transfect than primary cells. In addition, the nature of the DNA plasmid itself can affect the efficiency of the transfection.
“For each new combination of a cell line and nucleic acid, it is important to do optimization in order to achieve highest efficiency. This is independent from the reagent a researcher is using,” says Constanze Kindler, Ph.D., senior global product manager for Qiagen, which offers a range of transfection reagents for DNA. These include non-liposomal reagents Effectene and Attractene. “To transfect a GFP construct into a HeLa cell is therefore much easier than co-transfecting a primary cell with three different vector constructs. Efficiency in the first case can be up to 95% for the primary cells, and for a co-transfection in primary cells, a customer would be more than happy to achieve 10%.”
To compare transfection efficiencies between products, it is necessary to set up a side-by-side comparison using the same cell line, the same nucleic acid and the same conditions. One must also follow the protocol from the manufacturer, as protocol variation can affect results.
“The success of transfections [depends] on many different parameters. For every specific cell line and plasmid back bone, an optimal transfection protocol has to be worked out,” says Cordula Jany, Ph.D., international product manager for Roche Diagnostics GmbH. Roche Applied Science has introduced two new transfection reagents for delivery of DNA into eukaryotic cells. The X-tremeGENE Reagents, which are multi-component, high-performance formulations for transfection of a broad range of cells, claim an ideal balance between high efficiency and low cytotoxicity. [Is “ideal balance” too much of an opinion? Should we rewrite it to be more neutral?]
There are factors other than the transfection reagent that affect transfection efficiency. These include the presence of serum, contamination and the number of passages, as well as the quality and vector construct of the DNA to be transfected. Adding serum to media can boost cell growth but carries the potential of endotoxin contamination. Serum is usually obtained from random bleeding of animals used in meat production, which can result in lot-to-lot variability. Qiagen recommends testing a small lot of serum with a control cell line before gambling your experimental line on it. Future purchases should be made only from the same lot. Transfection can be performed without serum, but efficiency tends to be lower. Cells also can become contaminated when bacteria and mycoplasma grow in the cultures; contamination negatively affects the health of the cells and their ability to be transfected.
An often overlooked factor in the success of transfection is passage number. Ideally, the passage number should be less than 50. Cells that have been passaged too many times change growth behavior, morphology and transfectability. Transfection requires high-quality DNA that’s free of RNA, protein, genomic DNA and bacterial endosomes. Super-coiled plasmid DNA tends to give higher yields than linear DNA, but linear DNA often allows for more efficient integration into the cellular genome. The vector construct itself can be toxic to the cell if it expresses too much protein, or if the protein is toxic to the cell.
What about cytotoxicity?
For stable transfections, a small number of cell clones that have integrated the foreign DNA into their genomes is enough to proceed. In that case, there is usually no concern with cytotoxicity. Transfection into primary cells presents more of a challenge.
“I would recommend the following steps if a researcher encounters cytotoxicity after transfection,” says Kindler. “The first step is to optimize the ratio of DNA to transfection reagent, in case cytotoxicity is not due to cell culture issues or low quality of DNA and all controls worked fine.” Once the optimal ratio has been found, she advises reducing the number of complexes used on the cells or shortening the complexes’ incubation time to reduce toxic effects.
“There is always some cytotoxicity associated with transfection,” says Kurt R. Yardley, Ph.D.,
manager of product development, transfection technologies, for Life Technologies. “Some can be associated with less than ideal plasmid DNA preps that may carry over excess salts or endotoxins. Other sources are from using non-optimal amounts/ratios of lipids or DNA.” Life Technologies developed its Lipofectamine LTX reagent to balance potency and cytotoxicity.
“Researchers need to take into account that the highest efficiency may not correlate with the best experimental output. There are many commercial reagents on the market that claim high efficiency but also impart a high degree of cytotoxicity to the cell. As a result, analysis of particular cell signaling pathways may be compromised by the induction of apoptosis or cell stress responses that can lead to a misinterpretation of results,” says Scott Hayes, vice president of scientific operations for Mirus Bio. Mirus Bio reagents have been formulated for low toxicity. The company’s first transfection reagent, TransIT-LT1, has been a mainstay for researchers. The LT stands for “Low Toxicity.”
To electroporate or not?
Life Technologies also offers a system for transfection via electroporation, the Neon Transfection System. Electroporation works by creating an electric field of defined voltage and length of time in a buffered solution containing DNA and the cells you are transfecting. The electrical field opens pores in the cells, allowing the DNA to enter.
Electroporation is very versatile and works with most cell types with good efficiency. It requires larger amount of DNA than transfection, and may be performed on intact tissue. However, strong pulses can damage the cells, and some pores may become too large and fail to close afterwards.
It is also difficult to target the DNA with any accuracy, because the electropermeability of the membrane is not specific. The resulting ion imbalance can harm the cells.
Cationic lipids and friends
Outside the realm of electroporation, most transfection reagents are cationic lipids that form endosomes absorbed by the cell. The endosome releases its payload inside the cell.
Genlantis, a division of Gene Therapy Systems, bases all its reagents on cationic lipid formulations—with the exception of its NeuroFECT reagent. GenePORTER, neuroPORTER and BaculoPORTER are customized for DNA transfection to various types of cells and applications. NeuroFECT is the only polymer in Genlantis' lineup, and it is designed to transfect neuronal cells, specifically those too sensitive for lipid-based transfections.
“There are still many cells and cell types that remain stubborn to transfections, like some primary or neuronal cells, for example,” says Larissa Karnaoukhova, customer support representative at Genlantis. “All our transfection reagents come with protocols containing suggestions and instructions on how to optimize transfection reactions so that efficiencies are maximized and toxicity is minimized.”
Polyplus-transfection's reagent jetPRIME is a new-generation cationic reagent that forms positively charged complexes with DNA that enter into cells via endocytosis. The DNA/jetPRIME complex acts like a “proton sponge” in the endosome, absorbing its acidity. The resulting endosome swelling and rupture releases the nucleic acid cargo into the cytoplasm.
“Transfection using jetPRIME is very powerful; it uses much less reagent and less DNA than competitors. As a result, transfection is gentle to cells and cost-effective,” says Anne-Lise Monjanel, deputy CEO, marketing and communications manager for Polyplus-transfection.
When choosing a transfection reagent, it is important to make sure the reagent is optimized for your intended cell type. Many reagents are labeled for a broad range of cell types, but do your homework and make sure you know whether yours is covered. If working with sensitive primary cells, it is especially important to seek out a reagent that has a track record for your cell type and application. Efficiency problems may be cytotoxicity problems in disguise. Keep cells happy to minimize cytotoxicity.