by Caitlin Smith
Transfecting cells can be an optimization game of trial and error. Fortunately, recent advances in transfection reagents that are suitable for multiple cell types are easing the process of transfection for many researchers. Still, there are many factors involved in choosing which reagents are best: “Cell type, cytotoxicity, stability of transfection reagent and nucleic acid complex, cell culture conditions such as the presence of serum or animal-derived components, transfection efficiency, convenience, downstream analysis and cost per transfection,” notes Krishnan Allampallam, regional marketing manager at Qiagen. “To find a perfect transfection reagent that optimally satisfies all the above factors is not yet a reality. Researchers often pick a couple of transfection reagents that work under their laboratory conditions and optimize it for all their research needs.” Some of the advances in multipurpose reagents discussed here may help you with your transfections.
Faster, high-quality transfection protocols
While the perfect transfection reagent doesn’t exist, Qiagen is trying to develop reagents that excel in the areas in which they think researchers (doing both low and high throughput) most want improvements: high transfection efficiency, low toxicity, short and flexible protocols, and also amenable to automation. “One product that offers flexible protocols, low cytotoxicity and convenience is Attractene, the next generation lipid reagent,” says Allampallam. “Attractene is a nonliposomal lipid that enables highly efficient DNA transfection of adherent cells, including difficult-to-transfect cell types such as HaCaT, MonoMac6, and HCT116. It can be used for a range of applications, including transient or stable transfection, cotransfection of different DNA molecules, and shRNA vectors for gene silencing experiments using the Fast-Forward protocol.” Qiagen’s Fast-Forward protocols allow researchers to seed and transfect cells on the same day, enabling them to finish an experiment in one day instead of two, as with traditional protocols. Attractene can also be used both in the presence or absence of serum without changing transfection efficiencies. “With the growth of biopharmaceuticals, there is an increasing need of reagents that are free of animal-derived components,” says Allampallam. “Qiagen addresses these needs through products such as Attractene that are free of animal-derived components.”
Finding a multipurpose transfection reagent when more cell lines are being added to the thousands already existing is not easy. “One of the biggest challenges is to find a transfection reagent that will give high transfection efficiencies with no toxicity or off-target effects for all cell lines,” says Allampallam. “Another challenge is inter- and intra-experiment variations in factors that affect transfection. Standardized solutions such as Qiagen’s new FlexiTube siRNA Premixes, which allow high consistency between different experiments, are Qiagen’s attempt to reduce the number of variables.”
Integrating transfection in cell biology
While the process of transfection is crucial for many applications, in other areas it is important to remember that it is not a normal part of the cells’ biology. “It is critical to understand that transfection can potentially disturb the biological system being studied,” says Kevin Kopish, strategic marketing manager in cellular analysis at Promega. “Getting high expression may not accurately represent the biology that needs to be studied. Similarly, the toxicity of a reagent may influence the biology being studied.” Promega’s FuGENE® HD Transfection Reagent is a non-liposomal transfection reagent that works well in a wide variety of cell types, with a very low toxicity.
Kopish believes that the future holds a greater appreciation of the concept that more is not necessarily better, when it comes to expression. “We see a growing understanding that the act of transfection and transgene expression can influence biology, and I think researchers will be more mindful of this,” he says. “Second, 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.”
Transfecting stem cells and in vivo
Many transfection reagents are designed to introduce either DNA or siRNA into cells. Polyplus Transfection’s new jetPRIME™ is a polyvalent transfection reagent made to deliver both DNA and siRNA into many different types of adherent cells. The company is also working on the challenges of miRNA and stem cell transfection. “The field is lacking specific tools to study [miRNA-mediated gene expression] such as miRNA and anti-miR transfection reagents that do not affect miRNA-regulated pathways,” says Patrick Erbacher, CSO at Polyplus Transfection. Currently the most efficient ways to introduce nucleic acids into stem cells is infection by viruses, or by electroporation. “The biggest challenge would be to extend the versatility of a transfection reagent to stem cells. In the near future, transient expression of various transgenes in these cells could lead to their differentiation into diverse tissues for therapeutic purposes.”
Kurt Yardley, product manager at Life Technologies, believes that the biggest challenges in developing transfection reagents are finding reagents capable of effectively transfecting primary and stem cell types in situ or in vivo without adversely affecting the cells. “Research relies on effective and reliable models, and it has become increasingly apparent that the workhorse cell types 293, HeLa and others do not meet the criteria of accurately depicting the biology of an organism,” says Yardley. “Therefore, there has been a rapid increase in the use of primary and stem cell types for model systems.”
Life Technologies’ new release, Invivofectamine® 2.0, is a lipid-based transfection reagent capable of delivering siRNA in vivo to various cell types. “We have shown in vivo transfection of virtually every cell in the liver and all cell types that comprise the liver,” says Yardley. “We have also shown transfection of cells in the spleen, pancreas and kidney. We have a validated protocol for in vivo delivery to the liver and more protocols for other organs on the way. This reagent is non-toxic and capable of being used in a multiple dosing regime to maintain gene silencing for extended periods, months to years after the initial injection.”
Yardley says that the most exciting aspect of Invivofectamine® 2.0 is that not only can researchers assess gene function quickly in vivo, and validate targets for disease-related studies, but also it is now possible to effectively produce a knockout mouse in just a few days. "in vivo transfection has had a difficult and rocky start, and the validation of siRNA as a drug molecule has always relied on an effective delivery mechanism," says Yardly. "The majority of the work in this area has been gathered by Alnylam Pharmaceuticals and other large pharmaceuticals with the goal of commercial drug delivery, leaving the research use market to make do with reagents not specifically designed for in vivo use. Now there is a new generation of in vivo delivery reagents available to researchers that will allow a clearer picture of gene function by allowing the research to move from the culture dish to the whole organism level."
For the future, Yardley predicts that most research will use primary and stem cells, as well as whole animal model systems. "The next obvious step from primary cells in culture is whole animal model systems where gene function can be assessed in its true context and complexity," he says.