Scott Martin, Ph.D., team leader for RNA interference (RNAi) screening at the National Institutes of Health, Center for Advancing Translational Sciences, talks to contributing editor Tanuja Koppal, Ph.D., about recent trends in the use of different types of cells and reagents for screening drug targets and cellular pathways. While his group does not continually look to evaluate and replace cells and reagents being used in the lab, he mentions that there are certain deficient areas such as cell transfection and cell imaging that could benefit from new and improved reagents entering the market.
Q: What types of assays do you currently run in your lab?
A: We are a high-throughput screening lab serving the National Institutes of Health (NIH) intramural research program, and so we screen various approved projects that come through our door. We work on a dozen different projects each year, and each project has a few different screens embedded in it, which are mostly performed using cell lines. Many of those screens are cytotoxicity screens, where we are looking for cell vulnerability between different types of cancers using traditional luciferase-based cell viability readouts. We also work with our collaborators to look at cellular pathways of interest in areas such as cancer or immunology, using reporter assay systems in engineered cells. These assays are either luciferase-based readouts or fluorescencebased high-content imaging, the latter of which tends to yield much more information. We also perform numerous high-content cellular assays using host cells infected with virus-expressing green fluorescent protein (GFP) to identify host factors important to viral spread. In that context, we do screen a number of different viruses in our lab. There are other broader high-content phenotypic assays using fluorescently modified proteins that we use to monitor changes in expression or cellular localization.
Q: Have you worked with assays using stem cells?
A: We have worked with stem cells in the context of reprogramming and will soon be moving into projects related to cell differentiation. The challenge working with induced pluripotent stem (iPS) cells is that the experiments are much longer, on the order of several weeks, so they require more liquid handling and sometimes multiple rounds of transfection.
Q: What challenges do you run into with using primary cells?
A: We perform our assays mostly in cell lines, because they are easy to grow and harvest and because they are highly transfectable. When you use primary cells or cells in suspension, growth and transfection both become a problem. We recently performed a small-scale pilot screen using mouse primary neuronal cells in a model that is relevant to retinal injury and glaucoma. For this project we had to use Neuro- Mag, a neuron-specific transfection reagent that uses magnetic nanobeads for transfecting the primary cells. We added the primary retinal ganglion cells to these nanobeads complexed with small interfering RNA (siRNA) for screening and set the plate on a magnet for the duration of the experiment. The transfections worked beautifully and reproducibly in these cells, and some of this early work has already been published. So we do try to work with some of these difficult-to-use cells, but it’s certainly a challenge.
Q: Can this magnetic technology be used in cell lines as well?
A: With cell lines it’s usually easy to use lipid-based transfection reagents, so there’s no need to experiment with magnets and other less-used technologies. Also, on a cost basis, the lipid reagents are generally cheaper than alternate technologies. We have also used nucleofection reagents in 384-well format, primarily for T cells, and have found it difficult to optimize, given the number of protocols and buffers that are available. It also requires too many cells and is hard to optimize to a point where you get good transfection without much toxicity. It works well with cDNAs, but for RNAi screens it requires much more siRNA than is needed with other protocols.
Q: Are you continually looking for new ways and reagents to do your assays?
A: We pretty much stick with what’s working, unless it’s for a new type of assay. Especially with the basic cellular reagents, they all seem to work pretty well. However, we would surely evaluate reagents where things are less worked out. For example, reagents that can enhance fluorescent signals in imaging assays are worth looking at because that’s an inherent limitation. We are also more apt to try new things in areas that are deficient such as in primary antibody staining or where we are experiencing difficulties, like with transfections. Certain transfection reagents claim to be more amenable to certain systems than others, and we will certainly test those.
Q: Do you experiment and test different types of cell growth media, buffers, and matrices?
A: Most of the projects that come to us come with recommended protocols, and hence we don’t play much with growth media and cell culture reagents. However, in terms of protocols, going forward we plan to stop culturing our cells in antibiotics. This will alleviate any concerns regarding low-level contamination that goes undetected. We are also becoming more rigorous about scheduled mycoplasma testing and putting cells in quarantine once they enter our lab.
Cell identification is another big issue, where people are reporting data using cells when the cells are not what they claim to be. So every time we get new cells we will now test them for mycoplasma and then send them out for identity testing or short tandem repeat (STR) profiling. The cell testing services are becoming cheaper and more accessible, and it’s definitely something that everyone should start doing. Cell misidentification is a huge problem, and scientific journals are also going to be requiring this testing soon.
Q: What are you looking to invest your time and money in this year?
A: There are a few different things we would like to look at this year. Long noncoding RNAs (LncRNA) seem to be an area of upcoming interest, and vendors are starting to provide reagents and tools to probe LncRNA and the “dark matter” in the genome. We have also done some microRNA (miRNA) screens, and what’s really frustrating there is that the libraries are constantly being updated and it’s hard to keep up with the additions. However, the major crux of what we do is still siRNA screening, and we will be looking more carefully at the on- and off-target effects in these screens. Finally, something that is outside our current workflow but that we would like to do is pooled short hairpin RNA (shRNA) screens. The infrastructure needed to do arrayed shRNA screens is complicated and is not something we are set up to do. However, many of our projects are amenable to shRNA screens, and so we would like to assess some of the shRNA reagents. From a reagent perspective such screens are now becoming more practical and affordable, and vendors are also offering these screens as a full service.
Q: Are you interested in the 3-D cell cultures?
A: I would like to start exploring 3-D cell culture-based screening, although it is not very practical for a high-throughput lab like ours. However, we could certainly do some follow-up experiments in 3-D cultures, which would be more relevant. The traditional 2-D, large-scale cellular screens are fine, but in terms of predicting how the results translate in in vivo, they are certainly thought to be inadequate. It is routinely thought that 3-D cultures are more physiologically relevant, and we want to take a closer look at that. We have some groups here at the NIH that are screening small molecule libraries in 3-D cultures and looking at differences in responses in 2-D versus 3-D screens. It would be interesting to find out how different the data really is and if the differences in 3-D are in fact more clinically relevant.
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