Abstract
Secreted reporter molecules offer a means to evaluate biological processes in real time without the need to sacrifice samples at pre-determined endpoints. Here we have adapted the secreted bioluminescent reporter gene, Metridia luciferase, for use in a real-time viability assay for mammalian cells. The coding region of the marine copepod gene has been codon optimized for expression in human cells (hMLuc) and placed under the control of the human β-actin promoter and enhancer. Metridia luciferase activity of stably transfected cell models corresponded linearly with cell number over a 4-log dynamic range, detecting as few as 40 cells. When compared to standard endpoint viability assays, which measure the mitochondrial dehydrogenase reduction of tetrazolium salts, the hMLuc viability assay had a broader linear range of detection, was applicable to large tissue culture vessels, and allowed the same sample to be repeatedly measured over several days. Additional studies confirmed that MLuc activity was inhibited by serum, but demonstrated that assay activity remained linear and was measurable in the serum of mice bearing subcutaneous hMLuc-expressing tumors. In summary, these comparative studies demonstrate the value of humanized Metridia luciferase as an inexpensive and non-invasive method for analyzing viable cell number, growth, tumor volume, and therapeutic response in real time.
Highlights
High throughput cell viability and cytotoxicity assays are a mainstay for the biologic and therapeutic community
Cytotoxicity assays can overcome the need for cell lysis by measuring leaky dehydrogenase activity in the supernatant, which escapes through the damaged membranes of dead or dying cells [4,5]
The results confirm that serum does inhibit Metridia Luciferase (MLuc) activity but, contrary to previous reports [10], we reveal that hMLuc can be detected as an ex vivo reporter in mouse tumor model systems
Summary
High throughput cell viability and cytotoxicity assays are a mainstay for the biologic and therapeutic community. The resulting reporter vector, pDonor-hb-Actin-hMLuc, was used to generate stable transfectants in several human (C4-2, LNCaP, PC-3, HCT-116) and mouse (Tramp-C2) cell lines. Further studies with stable HCT-116-hMLuc colon cancer cells and transiently transfected HEK293 human embryonic kidney cells support the sensitivity and linearity of this reporter assay (Figure S1; Methods S1).
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