Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo

Abstract

Secreted reporters are a useful tool in the monitoring of different biological processes in the conditioned medium of cultured cells as well in the blood and urine of experimental animals. Described here is a protocol for detecting the recently established naturally secreted Gaussia luciferase (Gluc) in cultured cells as well as in blood and urine in vivo. Furthermore, the assay for detecting the secreted alkaline phosphatase (SEAP), the most commonly used secreted reporter in serum, is also presented. The Gluc reporter system has several advantages over the SEAP assay, including a much reduced assay time (1-10 min versus 1.5-2 h), 20,000-fold (in vitro) or 1,000-fold (in vivo) increased sensitivity and a linear range covering over five orders of magnitude of cell number. Additionally, the Gluc signal can be detected in urine and the signal can be localized in animals using in vivo bioluminescence imaging.

Figure 1. In vitro detection of secreted reporters

Different numbers of Gli36 human glioma cells expressing SEAP or Gluc were plated in wells of a 96-well plate and the conditioned medium was assayed for Gluc and SEAP after 24 h of culture (a). Cells (20,000) expressing Gluc were plated into 96-well plates and Gluc activity in the medium was assayed at different time points of culture (b). Results presented are mean ± s.d. (n = 5 with the experiment repeated 3x). RLU, relative light units. Figure adapted from Badr et al., 2007.

Figure 2. Blood monitoring of in vivo processes

Different numbers of Gli36 human glioma cells expressing both Gluc and SEAP were implanted subcutaneously into 5 athymic nude mice. After 48 h, 5 μl of blood and urine was assayed for Gluc and 5 μl of serum assayed for SEAP activity (a). Data presented as mean ± s.d. (n=5 with experiment repeated 3x). Gluc signal was localized in the animals using a CCD camera and in vivo bioluminescence imaging (b). Gli36 cells (1 × 10⁶) expressing the Gluc reporter were implanted subcutaneously into nude mice (n=10). On d 10 after implantation, tumors were injected with either PBS (control, n=5) or an HSV oncolytic vector known to kill tumors (n=5). Gluc reporter activity was monitored in the blood at different time points (c). C17.2 neuroprecursor cells (1 × 10⁶) expressing the Gluc reporter were injected i.v. into 5 nude mice and Gluc activity in the blood was monitored over time. Data presented in c-d are from one representative mouse from each group with the experiment repeated 3x. RLU, relative light units. Figure modified from Wurdinger et al., 2008.