Successful high-throughput profiling demands robust assays, minimal physical manipulation and homogeneity across the expressed target family of assays. When we design our assay platforms, we leverage our extensive assay construction experience to build cellular systems that incorporate numerous features to satisfy these requirements. In general we will deploy non-adherent cells with division rates less than 24 hours, this minimizes processing time for maintenance of the cells and initiating the assay. We also leverage techniques that manipulate cell division and/or programmed cell death; compounds that either directly or indirectly impact these pathways will impart a direct signal amplification due to the differential between unaffected proliferating cells and those that are growth arrested or dead. The power of this approach is best demonstrated in the time lapse images captured for one of our tyrosine kinase assay.
To detect affected cells, we incorporate a marker that can be readily detected with standard laboratory readers. These markers can include fluorescent proteins or enzymes such as luciferase. Our proprietary expression vector series are designed to express the target of interest, a detection marker, and a dominant selectable marker on a single bicistronic transcript. Each locus on the transcript typically encodes more than one functional domain constructed as a fusion protein, with multiple fusion proteins translated through the use of an intra-ribosomal entry site (IRES).
Finally, we use a single cell type when constructing our assay panels. This ensures a uniform genetic background between each assay, with the only differences contributed by the loci encoding the target and marker proteins. In this way, differential activities detected when profiling a compound are most likely attributed to it’s impact directly on the target protein, and less likely to be an indirect impact on an unrelated protein, which will be shared in common by each assay in the panel. Many of these cell types can be difficult to modify through traditional routes of transfection, for this reason, our expression vectors are designed to be transmitted by retroviral transduction, and the LTR has been carefully selected to express across a broad range of cell types.