A Firefly Luciferase Dual Color Bioluminescence Reporter Assay Using Two Substrates To Simultaneously Monitor Two Gene Expression Events

Abstract

Effective methods for monitoring eukaryotic gene expression and regulation based on bioluminescence - the emission of light by living organisms - are well established. Typically, the expression of a gene of interest is reported on with high sensitivity and over a wide dynamic range by the emission of light from a variety of engineered luciferase genes from beetles and marine organisms. The luciferase reporter genes are expressed downstream of the target gene or promoter and detected after exogenous addition of luciferin substrates. We describe a novel bioluminescence reporter method for the simultaneous monitoring of two genes expressing engineered firefly luciferase variants that emit readily distinguishable green and red light signals. The key feature is the selectivity of the enzymes for two luciferin substrates that determine each emission color. To validate our method, we performed a complex promoter transactivation experiment side-by-side with the Dual-Luciferase Reporter protocol and obtained essentially identical results. Additional comparative experiments demonstrated that our assay system provided improvements in background, cell normalization, and detectability compared to representative available methods. With access to a luminometer equipped with two optical filters, this method is an excellent choice for genetic reporter assays that can be performed with a single reagent solution.

Figure 1

Bioluminescence emission and detectability of PLG3 and PLR1 assayed with DSM. (a) Photograph of in vitro BL reactions of 1 µg PLG3 and PLR1 with DSM. Chemical structures of the benzothiophene (BtLH₂) and firefly (LH₂) luciferins responsible for each emission color are shown. (b) Simultaneous detection of mixtures of lysates from equal numbers of HEK293T cells transfected with pCMV-PLG3 or pCMV-PLR1 using a Synergy™ 2 microplate reader equipped with narrow bandpass filters. The relative light units (RLU) measured through the 516 ± 10 nm filter (blue line) and 635 ± 16 nm filter (red line) represent the activity expressed from the pCMV-PLG3 and pCMV-PLR1 plasmids, respectively. (c–d) BL emission spectra of lysates from equal numbers of HEK293T cells transformed with pCMV-PLG3 or pCMV-PLR1. The lysates were assayed (c) individually or (d) mixed together.

Figure 2

Comparison of the DART and Chroma-Glo™ Luciferase assay systems. Normalized spectral overlap measured with (a) DART and (b) Chroma-Glo™ using lysates from equal numbers of HEK293T cells transformed with (a) pCMV-PLG3 and pCMV-PLR1 assayed with DSM or (b) pCMV-CBG99 and pCMV-CBR assayed with Chroma-Glo™ reagent. Assays were performed using a Synergy™ 2 microplate luminometer equipped with the indicated filters. The relative emission from pCMV-PLR1 or pCMV-CBR detected through the 516 ± 10 nm filter (blue hatched bars) and the relative emission from pCMV-PLG3 or pCMV-CBG99 detected through the 635 ± 16 nm filter (red hatched bars) are shown. (c) BL emission from the same experiments detected through the indicated filters. The mean relative light units (RLU) ± STD for triplicate assays are depicted.

Figure 3

Comparison of the DART and DLR^® reporter methods. In separate experiments, HEK293T cells were co-transfected with pMEF3/TATA-PLR1 or pMEF3/TATA-Luc+ in the presence and absence of plasmids expressing Six4 and Eya2. The pCMV-PLG3 or pRL-CMV Lucs were included as the corresponding normalization controls. (a) After normalization to the pCMV-PLG3 or pRL-CMV activity, the fold reporter gene activation of pMEF3/TATA-PLR1 or pMEF3/TATA-Luc+ in the absence of both Six4 and Eya2 was set to 1.0. All reporter signals were expressed as the mean fold-amplification ± STD (duplicate transfections, assayed in triplicate). The normalized reporter activities were not significantly different (t-test) between the two methods when comparing MEF3/TATA alone (P = 0.97), in the presence of Six4 (P = 0.14), and in the presence of Six4 and Eya2 (P = 0.22). (b) The S/N of the reporter methods is represented by the ratio of the raw signals from pMEF3/TATA-PLR1 and pMEF3/TATA-Luc+ compared to those of the corresponding lysed untransfected cells (hatched bars). (c) Plots showing the simultaneous monitoring of the pCMV-PLG3 normalization control (black) and pMEF3/TATA-PLR1 reporter (red) signals using DART (with filters) to produce the normalized data illustrated with red bars in panel a.