This screening methodology is highly modular, consisting of a cDNA expression library, reporter construct, and assay cell line. Any of these can be substituted or modified to focus a screen on a biological target with particular properties.
The cDNA expression library
should be derived from a cell line or tissue that contains the desired activity and should contain as many full-length cDNAs as is possible. Many commercially available libraries are available and some have been characterized with regard to complexity (i.e. how many different clones or genes are represented) and frequency of full-length clones. Expression libraries can also be constructed from a particular source using available kits or protocols [13
]. In addition, whole-genome collections of cDNAs are now available from several commercial sources, so it is very possible to consider screening individual cDNAs or pools of cDNAs of known identity. A potential disadvantage of these whole-genome collections is that they may not contain particular tissue-specific splice variants of a gene that could encode a desired activity not conferred by other splice variants.
The complexity of pools being screened is another variable that can be adjusted. In our screen for proteins that signal to NF-κB, we used a pool complexity of 100 cDNAs/pool because pilot studies with the TRAF2 protein (an E3 ubiquitin ligase) had suggested that this complexity would allow detection of molecules several fold weaker than this protein of “average” potency in the assay. Pool complexity can be as small as one, i.e. an individual cDNA, and can be as large as is allowed by the sensitivity of the reporter assay, but the complexity will determine the minimum specific activity that can be detected in the screen. In these screens, transfection efficiency is usually optimal at a given concentration of total DNA used for the transfection. Since this parameter is held constant, say at 400 ng per 105 cells, the higher the complexity of that 400 ng of pool DNA, the lower the concentration of each assayed cDNA in the pool. A pool complexity of 100 cDNAs/pool will allow the assay of 4 ng each for each of the 100 cDNAs in the pool. In contrast, a pool complexity of 10 cDNAs/pool would allow 40 ng of each cDNA to be assayed, while a complexity of 1 would allow 400 ng of the cDNA to be assayed. It is not predictable how many ng of a particular cDNA need to be transfected to achieve detectability in the assay, and this parameter will be highly variable from cDNA to cDNA, and screen to screen. Higher-complexity pools allow the screening of more cDNAs per series of assays and therefore one confronts a trade-off between sensitivity and throughput that each investigator must judge.
The reporter used for screening offers many opportunities. It may consist of one or more copies of an element recognized by a particular transcription factor, and to a first approximation, the more copies of the element, the higher the sensitivity and dynamic range of the luciferase assay. Many reporters for transcription factors are in wide use and could be used for screening. The reporter may also contain a natural gene promoter, enhancer, or other regulatory element, and this screening methodology may be used for the study of an uncharacterized regulatory element. We have used luciferase-based reporters, but other modalities should be compatible with screening as long as their outputs can be quantitatively determined and normalized for transfection efficiency and extract recovery.
The cell line or tissue to be used should support the biological activity that is of interest, and should be as transfectable as is necessary to detect basal and induced reporter activity. We have routinely used HEK293T cells because of their ease of transfection and because they support all activities that we have pursued. Although these cells are derived from human embryonic kidney, they can support the activity of signaling proteins that otherwise are thought to operate in a tissue-specific manner. For example, HEK293T cells could support CARD11 activity when overexpressed, even though CARD11 is not expressed endogenously in these cells. Some activities, in contrast, may require the screen to use a particular cell type or tissue and may even require the use of primary cells.