In this interview Dr Robin Ketteler, who heads a research team at the Medical Research Council Laboratory for Molecular Cell Biology at University College London, discusses how the Cellaxess Elektra Discovery Platform has revolutionized their research on neuronal polarization.
Hi Robin, tell us a little more about your work in the Translational Research Resource Center
The Translational Research Resource Centre is a high-throughput screening facility that uses siRNA, cDNA and small molecule libraries to study cellular processes in cell-based assays. We are located in the middle of the University College London campus and our research interests cover diverse areas such as protein trafficking, autophagy, virus infection, cellular polarity, signal transduction and cell cycle progression.
You carried out an extensive validation of the Cellaxess Elektra platform. Could you tell us a little bit more about this?
In collaboration with the group of Dr. Antonella Riccio, we were interested in identifying genes that regulate neuronal polarity. While this was a major interest of her lab initially, there were problems in achieving high levels of transfection in primary neurons. With the Cellaxess Elektra, we were able to efficiently transfect plasmid DNA and siRNA oligonucleotides into primary rat cortical and hippocampal neurons. Since then, we have performed a siRNA screen using the rat druggable genome library and identified genes that regulate neuronal shape and morphogenesis. We have also tested the instrument for use in other primary cells including human macrophages and HUVEC’s and it is working very well in those cell types for transfection of plasmid DNA.
What methods of transfection are you currently using in your lab?
We are using various transfection methods in the lab, including calcium phosphate-based transection, lipid-based transfection methods and electroporation. The main determinants for selecting the right method are transfection efficiency, toxicity and cost. However, sometimes we see adverse effects in some techniques more than others – for instance, both calcium phosphate transfection and lipofection-based methods can induce a cellular stress response known as autophagy and in such cases, electroporation-based methods are much more beneficial.
What would you say are the main benefits of using an in-situ transfection system such as Cellaxess Elektra in your research?
Advances in biomedical research are driven by the development of novel technologies. Over the recent years, we have seen a surge in the development of novel cellular systems in the areas of primary cell culture and stem cell models that offer huge advantages over more widely used transformed cell lines. However, there have always been concerns in the applicability of these systems to large-scale genomic screening projects, partly due to difficulties associated with efficient transfection of these cells. One of our main focus areas is image-based high-throughput screening in primary cells, and for this we require an electroporation instrument that is easy to use, produces highly efficient transfection of plasmid DNA and works on adherent differentiated cell types. With the Cellaxess Elektra, we can deliver electrical pulses to cells without the need for suspension cultures, and without any adverse toxic effects. This will in turn enable several novel studies that were not possible in our lab a few years ago.
Which applications do you foresee in the future?
This is a particularly interesting time in genomic research and we are now at the crossroads of using physiologically relevant cell lines in large-scale automation.
Patient-derived induced pluripotent stem cells (iPSCs): Several novel technologies have emerged over the past years and these include the use of patient-derived iPSCs, as well as novel genome editing tools for the manipulation of cells. There are tremendous advantages in using patient-derived primary cells, as these can be differentiated into various lineages of interest such as neurons or cardiomyocytes. Many of these differentiated cells require the use of electroporation techniques in adherent cell lines, and the Cellaxess Elektra is extremely useful in this area.
Relevant cell models: Another area is the use of primary cell cultures in general. We believe that these cells offer a much better physiological relevance than classic transformed cell lines. In particular, the use of genetically modified animal models such as gene knockout and transgenic animals has huge advantages in studying gene function in a defined physiological model. The applications in this area could be two-fold: first, electroporation is commonly used to introduce the gene recombination cassettes into stem cells and with the Cellaxess Elektra this could be done on a much larger-scale than ever before. Second, cells from the genetically modified organism – such as fibroblasts from a knockout mouse – can be used in large-scale screening projects. Novel technologies such as the recently described CRISPR/Cas9 system for genome editing also rely on the introduction of a plasmid vector into cells and again, there is the potential to do this on a large-scale in primary cells.
Robin Ketteler studied biochemistry at the Free University Berlin and did his PhD at the Max-Planck-Institute for Immunobiology. After post-doctoral work at the Massachusetts General Hospital, Boston, in the lab of Brian Seed, Robin established the Translational Research Resource Centre at MRC LMCB in London in 2009. This academic high-throughput screening centre facilitates siRNA, cDNA and chemical screening in primary cells and cell lines. Main research insterests focus on autophagy, protein trafficking, mitogenic signaling and virus infection. Recently, with support from the Wellcome Trust, the facility has acquired the Cellaxess Elektra for high-throughput transfection of primary cells.