A High-Throughput Experimental Tool for Studying Gene Pathways

Introduction

The post-genome era has lead to great advances in high-throughput genomics studies. Genomic approaches such as microarray expression analyses and Chromatin Immuno-Precipitation on Chip (ChIP-chip) transcription factor binding assays have yielded new discoveries and insights into genetic pathways. These technologies provide valuable observational data but do not identify the functional connections within networks or explain the mechanism of gene regulation. Reporter gene based experiments like functional promoter assays provide an important additional layer of data for understanding mechanisms of regulation in genetic networks. Efficient and reproducible transfection methods are a prerequisite for all of these assays involving transfections.

An example of the power of combining multiple data types is shown in Figure 1. There are a number of points on the chart that fit with the prevailing wisdom that basal transcription factor binding (TAF250 in this case), functional promoter activity, and endogenous transcript levels will be correlated. However, it is the exceptions to this pattern that are the most interesting. Those loci with high endogenous transcript levels and promoter activity that do not show evidence of basal transcription factor binding are candidates for being TAF250 independent promoters. Likewise, those loci with significant transcription factor binding and strong promoter activity paired with low endogenous transcript levels are candidates for being regulated at the level of transcript turnover. Therefore, moving to a scale-up of functional promoter assays is very important, and our previous work in studying the function of hundreds of promoters across multiple cell lines using FuGENE® 6 Transfection Reagent was an important first step in the functional annotation of the genome (Figure 2).

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To facilitate high-throughput studies of regulatory element function, SwitchGear Genomics has produced libraries of thousands of human promoters encompassing many different disease-related pathways. SwitchGear’s regulatory element libraries are built using a luciferase reporter technology and are made available as transfection-ready tools to be combined with FuGENE® 6 Transfection Reagent for cell-based studies. These tools allow researchers to measure the function of thousands of regulatory elements in a single experiment. SwitchGear’s functional tools have successfully been used to identify new members of gene networks and have also been used as efficient and cost-effective screening tools to detail how compounds affect entire pathways rather than just a single marker. These new screening tools will also enable researchers to greatly enrich existing genomic datasets and provide functional annotation that will thoroughly describe the mechanism of action of a compound in a particular pathway. To ensure the success of high-throughput studies, consistent and efficient transfection conditions must be established for the experimental system. FuGENE® 6 Transfection Reagent provides the optimal transfection efficiency, low cytotoxicity, and reproducibility needed for high-throughput cell based assay studies.

Materials and Methods

HT1080 cells (ATCC® CCL-121™) were grown and seeded in Advanced MEM with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin. The cells were seeded at 3,750 cells/well in 100 µl total volume or 1,500 cells/well in 30 µl total volume (96- and 384-well formats respectively) in white tissue culture treated multi-well plates and incubated for 24 hours prior to transfection at 37°C in 5% CO2. For each 96-well transfection we mixed 1.67 µl experimental plasmid (30 ng/µl), 0.3 µl FuGENE® 6 Transfection Reagent, and 3.03 µl OptiMEM and incubated for 30 minutes at room temperature before adding to the well of seeded cells. For 384-well transfections, we conducted the same procedure with 1 µl plasmid (30 ng/µl), 0.12 µl FuGENE® 6 Transfection Reagent, and 1.88 µl OptiMEM.

For the control construct experiment, we incubated the transfected cells for 24 hours at 37°C in 5% CO2 before conducting the assay. For the hypoxia experiment, we incubated the transfected cells for 8 hours and then added DFO to a final concentration of 100 µM and continued incubation for 3, 6, 9, 24, and 34 hours before conducting the assay for luciferase activity. Each transfection and assay experiment was conducted with 4 replicate wells.

To assay luciferase activity in each well, we added 100 µlor 30 µl (96- and 384-well formats respectively) of Steady-Glo Luciferase Reagent directly to the well of transfected cells and incubated for 15 minutes shaking at room temperature. We then used the LMax384 Luminometer to read the luminescence signal from each well for 2 seconds. For the control panel, we normalized the raw data by the average activity of the random control constructs to yield promoter activity as a fold increase over the average strength of the random control construct fragments. For the hypoxia experiment, we calculated the fold induction in activity over that of the promoter at time-point zero.

Results and Discussion

Generating highly reproducible data is a necessity when scaling up cell-based functional promoter assays involving transfections. To demonstrate the reproducibility of the approach, we individually transfected a panel of 8 control constructs (four known promoters, four random genomic fragments each on a luciferase-based plasmid) into human tissue culture cells in 6 replicates in 384-well format utilizing the FuGENE® 6 Transfection Reagent and evaluated the behavior of the constructs. In the second study, in 96-well format, we transfected four independent preparations of a luciferase construct containing a promoter thought to be involved in the cell’s response to hypoxia (the LDHA promoter) and measured its activity across a timecourse after addition of DFO (a known activator of the hypoxia response).

Figure 3 shows that in 384-well format, the results of the functional promoter assay are highly reproducible with a low (and consistent) coefficient of variance (CV) associated with each of the eight constructs. This experiment also serves to demonstrate the large dynamic range of such an assay. Both the positive and random control fragments were chosen to represent the range of activity often observed among human promoters and randomly sampled genomic fragments, respectively. A comparable experiment in 96-well format yields similarly encouraging results (data not shown). Likewise, the hypoxia induction experiment shows that transfection-based functional promoter assays conducted in high-throughput format are also capable of measuring induction events over a time-course and show very consistent results between independent construct preparations and over a broad dynamic range (Figure 4).

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Conclusions

High-throughput  transfection-based functional promoter assays are an integral part of any genetic network or pathway study. The data shown here highlight the ability to gather highly reproducible data over a broad dynamic range for hundreds or thousands of promoters in a single experiment using FuGENE® 6 Transfection Reagent. Such large-scale experiments are made possible to many researchers by the availability of transfection-ready plasmids representing thousands of promoters from the human genome and the combination with an efficient, low cytotoxic, and highly reproducible transfection technology.

Fugene is a registered trademark of Fugent, L.L.C., USA.

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This article was originally published in Biochemica 4/2007, pages 26-28. ©Springer Medizin Verlag 2007