A robust dual reporter system to visualize and quantify gene expression mediated by transcription activator-like effectors

Abstract

Background: Transcription activator-like effectors (TALEs) are a class of naturally occurring transcription effectors that recognize specific DNA sequences and modulate gene expression. The modularity of TALEs DNA binding domain enables sequence-specific perturbation and offers broad applications in genetic and epigenetic studies. Although the efficient construction of TALEs has been established, robust functional tools to assess their functions remain lacking.

Results: We established a dual reporter system that was specifically designed for real-time monitoring and quantifying gene expression mediated by TALEs. We validated both sensitivity and specificity of this dual-reporter system in mammalian cells, and demonstrated that this dual reporter system is robust and potentially amenable to high throughput (HTP) applications.

Conclusion: We have designed, constructed and validated a novel dual reporter system for assessing TALE mediated gene regulations. This system offers a robust and easy-to- use tool for real-time monitoring and quantifying gene expression in mammalian cells.

Figure 1

Design and Construction of the dual reporters and their functional characterization. (a) Schematic representation of core features of the dual reporter system, depicting the two reporter genes GFP and luciferase separated by T2A (self-cleavage peptide), under the control of a minimal CMV promoter (mCMV). The multiple cloning sites (MCS) were incorporated to facilitate the insertion of TALE binding sequences. (b) Schematic representation of the promoterless dual reporter system serving as a negative control. (c) Schematic representation of the dual reporters under the control of a full CMV promoter serving as a positive control. HEK293 cells transfected with the negative control (promoterless) displayed weak GFP expression (d), while the minimal CMV promoter resulted in low to moderate GFP expression (e); the positive control (full CMV promoter) showed strong GPP expression (f). (g) Luciferase activities of each reporter, displayed in arbitrary units. Error bars indicate SD; n = 3. (h) Flow chart showing how to use the dual reporter to monitor TALE action. Specific TALE reporters can be constructed by inserting the TALE binding sequence (TBS) into the MCS. These customized reporters can then be used to trace TALE action in mammalian cells by co-transfection with the corresponding TALE.

Figure 2

Specific activation of dual reporters by TALEs. (a) Upper two panels: TALE-TF designed to activate the Sox2 gene were placed under the control of either CMV or EF1-a promoter. The target sites were selected from the 200-bp proximal promoter region of human Sox2 as indicated by Sox2-TALE binding sites (SBS). Lower two panels: the corresponding customized dual reporter was constructed by inserting 3xSBS. A control reporter harboring 3xUBS was set as a negative control. (b) Upper panel: Images of TALE-induced GFP reporter expression in trasnfected HEK293 cells. Lower panel: Levels of luciferase activities in transfected HEK293 cells, as determined by quantitative luciferase assay, in comparison to mock controls. Mock control cells received the same amount of plasmid DNA, but the Sox2 TALE-TF plasmid was replaced by empty vector. Data are expressed as mean ± SD, n = 3.

Figure 3

The robustness of dual reporters. (a and b, upper panels): Images of TALE-induced GFP reporter expression in HEK293 cells. (a) Low-range of Sox2-TALE-TF input, images taken at 20x magnification (b) High-dose range of Sox2-TALE-TF input, images taken at 10x magnification. (a and b, lower panels): Levels of luciferase activities expressed as mean ± SD, n = 3 at low-dose (a) and high-dose (b) range of Sox2-TALE-TF input. The fold induction of the reporter was calculated as relative activity of each dosage compared to the corresponding control (dual reporter plus empty TALE-TF vector).

Figure 4

Real-time monitoring and quantification of TALE in living cells. Images of TALE-induced GFP reporter expression in HEK293 cells were taken at indicated time-points after co-transfection of Sox2-TALE-TF and its dual reporter. (b) Fold induction of luciferase was calculated as relative activity at each time-point compared to its corresponding control (dual reporter plus empty TALE-TF vector). Levels of luciferase activities are expressed as mean ± SD, n = 3.