Bioluminescent reporters can be used to noninvasively detect and quantify biological processes in vitro and in vivo [1]. By engineering the reporter gene under the control of genetic promoters or other gene regulatory elements, the resulting reporter protein reflects the regulation of these elements. Examples of these regulatory elements can be constitutively active promoters to analyze cell survival and proliferation (e.g., CMV promoter) or specific promoters containing transcription factor regulatory elements (TREs) to analyze transcription factor activities [2, 3]. Other examples are 3′-UTR regions to analyze miRNA activities [4] and protease cleavage sites (PCSs) to analyze protease activities [5]. Regularly used bioluminescent reporter genes are Firefly luciferase [6] (Fluc) and Renilla luciferase [7] (Rluc). After translation these luciferases are localized inside the cell’s cytoplasm and ex vivo detection can therefore be a challenge. The enzyme Gaussia luciferase [8] (Gluc) is secreted into the extracellular fluids such as culture medium, animal blood, and urine and can therefore be readily noninvasively detected in these fluids enabling serial sampling over time in a single experiment.

One major disadvantage of luciferase reporters is the difficulty to use a multiplicity of luciferase reporters in combination in a single experiment. This limits the use of luciferase reporters in multiplex assays. Attempts are made to use a range of reporters using modified luciferases, each producing light with a different wavelength in order to discriminate between the different parameters they represent. The downside of this strategy is the need for sophisticated equipment to separate the different signals. Also, overlap in the light spectra might make signal separation a challenge and the number of reporters limited.

Another strategy to analyze multiple reporters in a single assay is to use luciferases that need different enzyme substrates to produce light [9]. A regularly used example is firefly luciferase, which needs d-luciferin as a substrate, in combination with Renilla luciferase, which needs coelenterazine. The disadvantage of this method is the limited number of luciferase substrate combinations currently known.

We aimed to develop an easily adaptable method for multiplexing a secreted luciferase reporter in order to perform broad range screening of biologically relevant processes in vitro or in vivo. Therefore, we engineered a range of fusion gene reporters consisting of the Gluc gene, each coupled to a different epitope tag [10] (see Fig. 1). By this method we engineered the ten Gluc_Tag reporters Gluc_Flag, Gluc_His, Gluc_HA, Gluc_AcV5, Gluc_V5 and Gluc_Glu, Gluc_Myc, Gluc_Kt3, Gluc_Au1, and Gluc_E2. Epitope tags [11] are peptide sequences usually derived from viral or bacterial genes, giving them a high affinity to mammalian antibodies. Coupling the Gluc gene to a range of different epitope tags enables us to design a large number of different Gluc_Tag reporters. Using the corresponding tag specific antibodies to selectively immunobind the different Gluc_Tag makes it possible to separate the different Gluc_Tag reporters and individually quantify them by reading out the light production after addition of substrate [10] (see Fig. 3). As a proof of concept we designed ten different Gluc_Tag based reporters, each coupled to a different epitope tag, under the control of the constitutively active cytomegalovirus (CMV) promoter. After lentiviral transduction [12] we produced ten U87 glioma cell lines, each stably expressing one Gluc_Tag reporter and used these cells to collect Gluc_Tag conditioned cell culture medium or mouse blood to monitor in vitro and in vivo tumor cell growth by measuring Gluc_Tag activity ex situ, after tag specific antibody immunobinding (see Fig. 3). In this chapter the proof of concept of the multiplex Gaussia luciferase-based functional reporter assays in vivo is described [10].