Editorial Article
Monday May 10, 2010
by Caitlin Smith
Introducing new genes into your cells, or silencing others, is easier each year as the technology and reagents for cellular transfection have steadily improved. And the improvement shows no sign of slowing down, spurred on by demands of users, according to Mary Kay Bates, senior scientist at Mirus Bio: “Exciting new advances in genomics and proteomics, stem cell technology, microRNA biology and analysis, and immunology and cancer research are driving the need for improved transfection efficiencies to better serve research and drug discovery efforts in these fields.”
The same basic problem of successful transfection remains for anyone wanting to change the genetic complement of their cells. “The biggest challenges to advancing transfection reagents today are much the same as those that we have always faced,” says Mike Vengrow, technical support manager at Genlantis, which offers both broad-spectrum and cell-type-specific transfection reagents based on cationic lipids and polymers. “Cells have evolved barriers to entry of foreign molecules. We have to identify methods and materials to circumvent these barriers.” Here are some tips and tools from transfection technology experts to help you with your own experiments.
Consider the reagent for your cell type
The different types of reagents may seem dizzying at first, but most agree that one of the most important first steps in choosing a reagent is to research your particular cell type and culture conditions. “It is important to scan the literature to discover which transfection methods worked for other researchers working with a particular cell type,” says Bates. “Different culture conditions and transfection formulations will affect the end results, so it is important to compare several different transfection reagents.”
Some reagents are better at transfecting most work-horse cell lines used in culture, while others might be better at transfecting specific cell lines. Roche’s FuGENE® HD and FuGENE® 6 Transfection Reagents are examples of reagents that can successfully transfect most cell types. “FuGENE® 6 Transfection Reagent offers slightly lower cytotoxicity than FuGENE® HD Transfection Reagent and is robust enough to transfect probably 90% of cell lines and types; FuGENE® HD Transfection Reagent is a little more aggressive, so it is able to transfect most of the remaining difficult-to-transfect cell lines/types,” says Mike Leous, group marketing manager for Roche Diagnostics. “The other application in which FuGENE® HD Transfection Reagent excels is in delivering higher yields of expressed protein.” A study of many reagents found that the FuGENE® reagents were best at transfecting embryonic stem cells1. Another recent study found that FuGENE® had the least off-target effects2. “The most exciting development we have seen is the benefit of minimizing unintentional up- or down-regulation of unrelated genes caused by the reagent itself—not by the transfected DNA,” agrees Leous. Invitrogen/Life Technologies makes another well-known reagent, Lipofectamine 2000, which also transfects a wide variety of commonly-used cell types.
Unfortunately, today there is no one reagent that works for every cell type, as cultures can vary considerably. “Given the diversity of cell types, it is doubtful there shall ever be a single transfection method that can be applied across all cultures,” says Peyton Hughes, VP of marketing at GeneCopoeia. “Optimization experiments should consider transfection efficiency versus toxicity, off-target effects, cell viability, and reproducibility. The type and quality of molecule (RNA, DNA shRNA etc.) as well as the passage number and quality of cells to be transfected play important roles in the overall transfection results.” GeneCopoeia’s EndoFectin™ reagent is a proprietary cationic polymer formulation that also works well for a wide variety of cell lines.
Rare cell types or primary cultures can still be a lot trickier to transfect, however. “The transfection challenges remain with slow-growing or non-dividing cell types such as primary neurons,” says Bates. “There are few currently available reagents that are able to efficiently deliver nucleic acids to these cell types.” Mirus Bio’s TransIT®-2020 Transfection Reagent, is a new animal-free reagent that may help. According to Bates, it “provides outstanding transfection efficiency in cell types that are commonly recalcitrant to chemical transfection, including many primary cells and stem-cell-derived cells.”
Consider expression level and toxicity
A major factor to consider is – for each cell type and nucleic acid – how much of the reagent in question would you require to achieve enough expression without incurring toxicity. “Researchers should consider the effects of toxicity when evaluating transfection reagents,” says Bates. “While high expression is clearly paramount, it is also important to consider how the reagent itself affects the physiologic relevance of the transfection results. In addition to measuring the expressed protein, it is vital to include proper controls, including untransfected cells, cells treated with the reagent alone and cells transfected with a positive control.”
“We see a growing understanding that the act of transfection and transgene expression can influence biology,” says Kevin Kopish, strategic marketing manager for cellular analysis at Promega, which sells Roche’s FuGENE® transfection reagents. “The fact that good transfection methods allow studies in cells such as primary cells will bring much more insight and tool development into what works well in these cells. For instance, standard viral promoters that drive transgene expression seem to function very differently in primary cells. This will require different promoters to be used, but can also aid in understanding gene expression through pathways such as epigenetic modulation.”
Qiagen’s latest release is designed for ease and speed. Their nonliposomal lipid Attractene Transfection Reagent “is our next-generation lipid reagent, that combines the features of our Effectene Transfection Reagent, like highly efficient DNA transfection and lowest cytotoxicity, with our new flexible Fast-Forward Protocols,” says Constanze Kindler, senior global product manager for transfection at Qiagen. “In Fast-Forward Protocols, cells are seeded and transfected on the same day.”
New transfection tools for siRNA
If you want to transfect with siRNA, more new reagents await you. Transfection reagents for siRNA remain particularly challenging. “There doesn’t seem to be a silver bullet transfection reagent for transfecting siRNA, so we are seeing more people using shRNA for knockdown, because FuGENE® HD and FuGENE® 6 Transfection Reagents work better with those than with siRNA,” says Leous. Some companies are trying to fill this siRNA niche, such as Polyplus Transfection, which plans to launch a new siRNA reagent that “permits the use of small amounts of reagents at low siRNA concentration, especially well-suited for screening applications,” says Patrick Erbacher, CSO at Polyplus Transfection. “Polyplus Transfection recently launched jetPRIME™, a new polyvalent transfection reagent for efficient delivery of DNA and siRNA across many adherent cell types.”
Qiagen also offers their HiPerFect Transfection Reagent for siRNA transfection. “It is a unique blend of cationic and neutral lipids that enables effective siRNA uptake and efficient release of siRNA inside cells, resulting in high gene knockdown even when using low siRNA concentrations,” says Joerg Dennig, global product manager for siRNA at Qiagen. “Research suggests that off-target effects, which may produce misleading results in RNAi experiments, can be largely avoided by using low siRNA concentrations.” Also, Qiagen’s FlexiTube siRNA Premixes contain stabilized complexes of siRNA and transfection reagent at a pre-optimized ratio, and can be stored for convenience.
The range of reagents available will likely continue to broaden steadily. As Bates says, “with [the] increasing emphasis on new drug discovery and emerging biotherapeutics, the need for efficient nucleic acid delivery to cells and tissues for expression studies and recombinant protein production will continue to drive advances in transfection.”
References
1Video: Efficient Gene Delivery to Human Embryonic Stem Cells, Dr. Kouichi Hasegawa, Institute for Frontier Medical Sciences
2Jacobsen LB, et al., "Transcriptional effects of transfection: the potential for misinterpretation of gene expression data generated from transiently transfected cells," BioTechniques 47: 617-624, 2009.