A critical assessment of the factors affecting reporter gene assays for promoter SNP function: a reassessment of -308 TNF polymorphism function using a novel integrated reporter system

Abstract

One of the greatest challenges facing genetics is the development of strategies to identify functionally relevant genetic variation. The most common test of function is the reporter gene assay, in which allelic regulatory regions are used to drive the expression of a reporter gene, and differences in expression in a cell line after transient transfection are taken to be a reflection of the polymorphism. Many studies have reported small differences in single nucleotide polymorphism (SNP)-specific reporter activity, including the tumor necrosis factor (TNF) G-308A polymorphism. However, we have established that many variables inherent in the reporter gene approach can account for the reported allelic differences. Variables, such as the amount of DNA used in transfection, the amount of transfection control vector used, the method of transfection, the growth history of the host cells, and the quality and purity of DNA used, all influence TNF -308 SNP-specific transient reporter gene assays and serve as a caution for those researchers who apply this method to the functional assessment of polymorphic promoter sequences. We have developed an integrated reporter system that obviates some of these problems and shows that the TNF G-308A polymorphism is functionally relevant in this improved assay, thus confirming that the -308A allele expresses at a higher level compared with the -308G allele.

Figure 1

Distribution of Luciferase expression from 18 independent experiments of −308A normalized to −308G and TwoStep cluster analysis. (a) Graphical representation of the fold difference in the transcriptional activity of −308A over −308G. Jurkat cells were transiently transfected using either −308G/GL3Luc or −308A/GL3Luc DNA and the cell lysates were harvested after 24 h. Luciferase activity was determined using the Dual-Luciferase reporter assay system (Promega). Each value (n=18) represents the mean fold difference in luciferase activity of −308A/GL3Luc over −308G/GL3Luc, for which each experiment was performed in triplicate. (b) The percentage and mean of the data values from panel a clustered in two groups. Initially, the TwoStep Clustering Component of the SPSS Statistical Analysis software package was used to automatically determine whether the ideal number of clusters was two, using both the Bayesian Information Criterion (BIC) and the Akaike Information Criterion (AIC).

Figure 2

Titration of −308G/GL3Luc and −308A/GL3Luc DNA with or without the human TNF untranslated region (3′UTR). Jurkat cells were transiently transfected with varying amounts of reporter DNA (5, 3, 1, 0.5, and 0.1 μg) and harvested after 24 h to assay for luciferase activity using the Dual-Lucifrase reporter assay system (Promega). Reporter plasmids that were used include (a) −308G/GL3Luc and −308A/GL3Luc without 3′UTR or (b) −308G/GL3Luc and −308A/GL3Luc in context of the 3′UTR. Experiments were performed in triplicate. Values represent fold difference of raw luciferase relative light units (RLU) from −308A/GL3Luc over corresponding −308G/GL3Luc.

Figure 3

Effects of co-transfection with pRL-TK control plasmid. Represents the fold difference in transcriptional activity of −308A/GL3Luc and −308G/GL3Luc after normalizing RLU/s to −308G/GL3Luc only, which was set at 1. Jurkat cells were transiently co-transfected with biallelic −308 TNF reporter constructs along with different amounts of pRL-TK (30, 100, 300, 500 ng, and 2 μg) and harvested after 24 h to assay for luciferase activity using the Dual-Luciferase reporter assay system (Promega). The experiment was performed twice in triplicate. Values represent fold difference of raw luciferase RLU/s from −308A/GL3Luc over corresponding −308G/GL3Luc. Statistical significance was determined by Student's unpaired t-test. P<0.05 was considered significant.

Figure 4

Differential expression is abrogated with transfection, time in culture, and age of DNA. Represents the fold difference in transcriptional activity after normalizing luciferase RLUs/ to −308G/GL3Luc, which was set at 1. (a) Jurkat cells were transiently transfected with either −308G/GL3Luc or −308A/GL3Luc DNA and harvested after 24 h to assay for luciferase activity using the Dual-Luciferase reporter assay system (Promega). Transfections were performed using Lipofectamine 2000 (Lipid-mediated; Invitrogen) or by electroporation as indicated. The experiment was performed thrice in triplicate. (b) Jurkat cells were cultured for 1, 2, or 3 weeks before transfection. The experiment was performed in triplicate. (c) (i) Represents three consecutive experiments performed in triplicate using DNA prepared from the QIAfilter Endofree Plasmid Maxi Kit (Qiagen) earlier and stored at −20°C. (ii) Represents the next three consecutive experiments performed in triplicate using fresh unstored DNAs prepared from the QIAfilter Endofree Plasmid Maxi Kit. The average fold differences of −308A/GL3Luc over −308G/GL3Luc from all experiments after normalization of raw luciferase RLU/s values to −308G/GL3Luc are shown. Statistical significance was determined by Student's unpaired t-test. P<0.05 was considered significant.

Figure 5

Comparative analysis of polymorphic −308 TNF promoter sequences. (a) Representation of both the −308G TNF and −308A TNF allelic promoter/GFP reporter constructs. (b) Both reporters were integrated into the FRT site of the FRT-Jurkat line and GFP expression assessed by flow cytometry. For cells carrying the −308G reporter, 34.5% of the cell population expresses GFP, whereas for the −308A reporter, 79.3% of the cell population expresses GFP. MF, mean fluorescence.

Figure 6

Cell cycle contribution to −308 allelic expression differences in the −308G and −308A FRT-Jurkat reporter cell lines (a and b). Dot plots of GFP fluorescence on y axis vs Propidium Iodide (PI) staining on the x axis to show sub-populations of cells in each phase of the cell cycle. G1, S, and G2M DNA contents are as labeled for each gate, with the percentage of GFP+ cells in each population shown in superscript. Panel a represents cell cycle analysis of the −308G reporter cell line, whereas panel b represents the −308A reporter line. (c) Graphical representation of seven independent cell cycle experiments of the ratio of the mean fluorescence (percentage of GFP+ cells multiplied by the mean channel fluorescence) in each cell cycle phase. Statistical significance was determined by Student's unpaired t-test. P<0.05 was considered significant.