Luciferase Reporter Assays to Assess Liver X Receptor Transcriptional Activity

Abstract

Luciferase reporter assays are sensitive and accurate tests that enable the analysis of regulatory sequences, the magnitude of transcriptional activity by transcription factors, and the discovery of gene regulatory elements and small-molecule modulators with high levels of precision. This is made possible through detection of bioluminescence produced by luciferase-coding reporters in a wide range of cellular environments. These assays are routinely used to analyze the activity of transcription factors, including the lipid-activated liver X receptor (LXR), in response to different stimuli as well as for the identification of their ligands. In this chapter we describe in detail the assays performed to investigate LXR activity in a macrophage-like cell line (RAW 267.4). These can be easily adapted to other nuclear receptors and transcription factors.

1 Introduction

Luciferase reporter assays are generally performed to (a) identify transcriptional regulatory elements (in promoters, enhancers, or untranslated regions), which control the expression of a gene or genes, (b) help identify ligands for ligand-activated nuclear receptors , and (c) examine changes in the activity of a particular transcription factor/nuclear receptor upon treatment with specific stimuli. Reporter assays rely on the regulatory element or region of interest being fused to a reporter gene not usually expressed in the system being tested, which when translated can be assayed or detected accurately. The level of detected reporter protein is thus directly proportional to the level of mRNA transcribed.

Reporter proteins to date have included β-galactosidase, chloramphenyl acetyltransferase, β-glucuronidase, secretory alkaline phosphatase, and a variety of different fluorescent proteins. One of the most widely used reporters is the luciferase gene, which encodes for the enzyme luciferase that oxidizes d-luciferin in the presence of ATP, oxygen, and Mg²⁺, to yield a product that can be quantified by measuring the released light. The luciferase assay has advantages over other reporter assays due to its high sensitivity, wide dynamic range, and relative affordable cost. This assay was first described in 1987 by de Wet et al. [1] and subsequently commercial kits have become widely available.

Liver X receptors (LXRs) belong to the nuclear receptor superfamily of ligand-activated transcription factors. LXRs are expressed as two isoforms: LXRα, which exhibits tissue- and cell- specific expression in liver, adipose, kidney, adrenal tissues, and macrophages and the ubiquitously expressed LXRβ [2]. LXRs heterodimerize with the retinoid X receptor (RXR) to transcriptionally modulate target genes involved in several processes, primarily in lipid metabolism and inflammation in which innate immune cells such as macrophages have been shown to play a key role [2]. We have optimised the luciferase reporter assay to measure nuclear receptor transcriptional activity and have used this technique successfully to examine gene regulation and identify target genes of different nuclear receptors (including LXR ) in a variety of cell lines [3–5]. The general strategy is to (a) identify the putative regulatory element in the gene of interest, (b) clone this putative regulatory element or the portion of a promoter/enhancer containing the regulatory element to a reporter gene (luciferase in this case), (c) transfect or incorporate reporter and expression plasmids into cells, (d) activate nuclear receptor with specific ligands, and (e) detect transcriptional activation by quantifying the luminescence with a luminometer.

In this protocol we describe how in less than a week, a researcher is able to transfect their previously cloned and produced plasmid construct (containing putative or already established regulatory elements together with constructs expressing LXR nuclear receptors) into the RAW macrophage cell line and detect the regulation of their putative regulatory elements using the luciferase reporter assay and β-galactosidase assay as a control. These cells represent an interesting option to study the differential effects of LXRα and LXRβ since they only express the beta subtype [6]. Overall, the assay is as follows: (a) cell transfection with luciferase and expression plasmid constructs, (b) cell lysis, (c) addition of luciferase substrate (d-luciferin), and (d) quantification of luminescence .

2 Materials

Prepare all solutions using ultrapure water (prepare by purifying deionised water to attain a sensitivity of 18 MΩ cm at 25 °C) and analytical grade reagents. Prepare and store all reagents at 4 °C or −20°C as indicated (unless otherwise noted). Diligently follow all local waste disposal regulations when disposing of waste materials.

Store commercially obtained reagents as advised in product manual.

2.1 Cell Culture

• 1. RAW 267.4 macrophage cell line (established from murine tumours induced by the Abelson leukemia virus, ATCC^®, TIB-71™).

• 2. Cell culture plates (100 × 20 mm) (sterile, wrapped in a sleeve of 10).

• 3. 24-well cell culture plate (sterile, individually wrapped).

• 4. Aspiration polysterene pipette (sterile, individually wrapped).

• 5. Individually wrapped polysterene serological pipettes, 25, 10, 5 mL.

• 6. Sterile PBS 1× (see Note 1 ).

• 7. Fetal bovine serum, stored at −20 °C.

• 8. Gentamycin (10 mg/mL stock), stored at 4 °C.

• 9. Growth medium: DMEM supplemented with 10 % FBS and 20 μg/mL gentamycin, stored at 4 °C.

• 10. Hemacytometer/cell counter.

• 11. Cell scraper/lifter with 19 mm blade, sterile and individually wrapped.

• 12. Gilson single channel pipettes and sterile tips.

• 13. Repeat pipettor.

• 14. Repeat pipettor tips, sterile and individually wrapped.

• 15. Incubator (37 °C, 5 % CO₂).

• 16. Tissue culture Class II cabinet.

• 17. 1.5 mL microcentrifuge tubes, sterile.

• 18. Sterile polypropylene 15 and 50 mL tubes.

• 19. Vortex.

• 20. Centrifuge with rotors for 1.5, 15, and 50 mL tubes.

• 21. LXR activators, i.e., GW3965 [7]/vehicle controls, stored at −20 °C.

• 22. Ice.

• 23. Luciferase Reporter Assay Kit including Luciferase substrate and Lysis buffer 5×.

• 24. Plate rocker. Water bath at 37 °C.

2.2 Luciferase Reporter Constructs and Transfection

• 1. Transfection reagent (TurboFect, Fermentas #R0531), kept at 4 °C or on ice (see Note 2 ).

• 2. Endotoxin-free preparations of plasmids diluted at required concentration (see Note 3 ).

• 3. OPTI-MEM^®I (Gibco, Invitrogen #S1985-026), stored at 4 °C (see Note 4 ).

• 4. Dulbeco’s Modified Eagles Medium (DMEM) (see Note 5 ).

2.3 β-Galactosidase Assay

• 1. o-Nitrophenyl-beta-d-galactopyranosidase (ONPG) 4 mg/mL⁻¹ in Buffer Z pH 7.5, stored at −20 °C and protected from light (see Note 6 ).

• 2. Buffer Z (Na₂HPO₄ 0.1 M, KCl 10 mM, MgSO₄ 1 mM), filtered and stored at 4 °C.

• 3. β-Mercaptoethanol.

2.4 Bioluminescence Detection

• 1. Luciferase reporter gene assay system (see Note 7 ).

• 2. White opaque 96-well microplates for luciferase reading.

• 3. Clear 96-well microplates for β-galactosidase reading.

• 4. Luminometer (see Note 8 ).

3 Methods

Carry out all procedures at room temperature unless otherwise specified.

3.1 Cell Seeding to 24-Well Plates

To be done 16–24 h before cell transfection under sterile conditions.

• 1. Wash previously cultured RAW 267.4 cells with 37°C pre-warmed PBS 1× using a serological pipette.

• 2. Discard PBS 1× with an aspiration pipette attached to a vacuum line.

• 3. Add 37 °C pre-warmed complete DMEM.

• 4. Harvest cells by scrapping them off the plates with a cell scraper.

• 5. Resuspend cells to a single-cell suspension (see Note 9 ).

• 6. Count cells with a hematocytometer to calculate cell density (cells/mL).

• 7. Calculate volume of cell homogenate required to get 2 × 10⁵ cells/well × number of wells required. Complete with growth medium to total volume calculated as 500 μL per well × total number of wells.

• 8. Seed 2 × 10⁵ cells (i.e., 500 μL of cell suspension) per well in a 24-well plate (see Note 10 ).

• 9. Incubate cells at 37 °C, 5 % CO₂, overnight.

3.2 Cell Transfection

Perform cell transfection procedure under sterile conditions.

• 1. Calculate amount of plasmid required to perform assay (see Note 11 ). Use 1.5 mL tubes for the DNA mixes, or for more than 3 ml of total volume use 15 mL tubes. All tubes should be sterile.

• 2. Pre-warm PBS, OPTI-MEM^®1 and complete DMEM at 37 °C.

• 3. For each well, prepare a DNA mix as follows (see Note 12 ):

• 4. In 50 μL total volume add all plasmids needed: 3 μL luciferase reporter vector (100 ng/μL), 1 μL LXR expression vector (i.e., pcDNA3-LXRa) at 50 ng/μL (or empty pCDNA3 vector as negative control) and 1 μL pCMV-Bgal (100 ng/μL). Complete with OPTI-MEM^® I (45 μL).

• 5. Mix by briefly vortexing to mix well.

• 6. Centrifuge briefly to collect sample at the bottom of the tube.

• 7. For each well, prepare a TurboFect mix as follows:

• 8. Add 1 μL of TurboFect to 49 μL OPTI-MEM^®I per well.

• 9. Mix gently by pipetting mix up and down (do not vortex).

• 10. Add TurboFect mix on top of the DNA mix (see Note 13 ).

• 11. Mix gently by pipetting up and down about 10×.

• 12. Incubate at room temperature for 20 min.

• 13. Before addition of the polymer-DNA mix to the cells, wash cells with pre-warmed PBS 1×.

• 14. Discard PBS 1× with an aspiration pipette attached to a vacuum line.

• 15. Add 900 μL of pre-warmed growth medium with a pipette repeater to one side of the well avoiding disruption of the cell layer.

• 16. Add 100 μL of DNA/TurboFect mix in the corresponding well a drop at a time.

• 17. Mix the growth medium with the added DNA/TurboFect mix by tilting the plate back and forth and left to right about 5–10 times.

• 18. Incubate cells at 37 °C, 5% CO₂, for 24 h.

3.3 LXR Activation

• 1. Calculate quantity of LXR activator and control solution required for a final volume of 500 μL per well (see Note 14 ).

• 2. Prepare activation/control solution with pre-warmed growth medium. Mix well.

• 3. Discard transfection medium with an aspiration pipette attached to a vacuum line.

• 4. Wash each well with 200 μL of pre-warmed PBS 1× (optional).

• 5. Add 500 μL of activation/control medium as prepared above with a pipette repeater to one side of the well avoiding disruption of the cell layer.

• 6. Incubate cells 18 h, 37 °C, 5 % CO₂.

3.4 Luminescence (LXR Activity) Detection

This step no longer needs to be performed under sterile conditions.

Follow the manufacturer’s instructions of the specific detection/assay system used. These are some general guidelines.

3.5 Luciferase Assay

• 1. Take out lysis buffer (usually a 5× or 10× stock stored @ −20 °C) at RT and thaw completely.

• 2. Prepare enough 1× lysis buffer with 18 MΩ water.

• 3. Take cell culture plates from the tissue culture incubator to RT.

• 4. Remove medium from cells with an aspiration pipette attached to a vacuum line (see Note 15 ).

• 5. Wash cells once or twice with 300 μL ice-cold PBS/well using a repeater and pipetting gently to one side of the well avoiding disruption of the cell layer.

• 6. Remove PBS (see Note 15 ).

• 7. Add 100 μL lysis buffer 1× (kept at 4 °C from 5× stock).

• 8. Lyse cells directly on the culture plates by placing them on a rocker at RT for 15–30 min (see Note 16 ).

• 9. Prepare luciferin substrate reagent, stock typically kept at −20 °C if not reconstituted.

• 10. Remaining luciferin substrate reagent can be stored at −80 °C till the next use.

• 11. Keep cell culture plates with lysed cells on ice for immediate reading or store plate with lysate at 4 °C until detection on the same day. (Alternatively store at −80 °C for detection on a different day).

• 12. Transfer 10–20 μL lysate from each well of the culture plate onto a white opaque 96-well plate (see Note 17 ).

• 13. Set up luminometer to inject 50 μL/well substrate (see Note 18 ).

• 14. Set detection for 5 s (see Note 18 ).

• 15. β-Galactosidase assay.

• 16. Prepare Buffer Z mix (80 % buffer Z + 20 % ONPG + 3.4 μL β-mercaptoethanol per mL of mix Z).

• 17. Transfer 20 μL lysate/well to a clear 96-well plate (see Note 19 ).

• 18. Add 200 μL of Buffer Z.

• 19. Incubate at 37 °C until colour change (yellow) becomes apparent.

• 20. Measure absorbance at OD 405 nm in a plate reader with the appropriate filter.

The transcriptional activity of LXR is calculated as luminescence units normalized to β-galactosidase reading.

4 Notes

• 1. Store at RT unopened, and once opened store at 4 °C. Pre-warm to 37 °C in a water bath before use.

• 2. In our experience with transfections in RAW cells, the type of transfection reagent is crucial. We have routinely and successfully used Turbofect, which is not a cationic lipid-based formula but a sterile solution of a cationic polymer in water. This polymer forms positively charged complexes with plasmid DNA that are stable and protect the DNA from degradation, thus facilitating efficient introduction of the plasmids into cells.

• 3. In our experience, endotoxin-free plasmid preps get transfected at a higher efficiency compared to standard DNA preparations. In order to minimize the volume of DNA over the total transfection volume required, we typically prepare working dilutions of all plasmids at concentrations high enough so that small volumes are used. Typically the plasmid needed are: (1) a luciferase reporter vector with either an LXR response element (LXRE) or a promoter/enhancer containing an LXRE fused to a luciferase cassette, (2) an expression vector with the LXR cDNA cloned, and (3) an expression vector containing β-galactosidase or a different luminescence reporter such as Renilla to be used as a transfection efficiency control. If changes in LXR activity by cofactors are being tested, additional expression vectors for those cofactors would be needed.

• 4. OPTI-MEM^®I is a special Modified Eagle’s Medium (MEM)-based media with components that allow for a reduction in FBS supplementation with no change in cell growth rates. It is also recommended for use with some cationic lipid transfection reagents. We typically use the version that contains a stable form of glutamine.

• 5. Other media such as RPMI can be used instead.

• 6. ONPG is the preferred colorimetric substrate to examine β-Galactosidase reporter activity. The product formed is soluble and has a high extinction coefficient at 405 nm. This substrate yields a yellow product that is easily detectable in the visible range. β-Galactosidase reporters are usually driven by a variety of promoters including the cytomegalovirus (CMV) promoter. To be used as a transfection efficiency control researchers will need to ensure that the activity of the promoter used remains unchanged with the experimental conditions (addition of LXR ligands and LXR ectopic expression in this case).

• 7. We routinely use commercial kits to analyse luciferase activity which typically include (a) a cell lysis buffer that allows luciferase and β-galactosidase or Renilla assays to be performed from the same extract, (b) a substrate solution, and (c) a buffer to dissolve the substrate. These solutions and buffers can also be easily prepared by the researcher.

• 8. Must be highly sensitive and able to detect a broad dynamic range of bioluminescence. Microplate options are preferable to single tube models if a large quantity of samples need to be assayed.

• 9. Mix cell suspension gently by pipetting up and down against the bottom of the tissue culture dish approximately ten times, do not vortex, and try to avoid generating bubbles.

• 10. To avoid pipetting errors this is best done using a repeater pipette.

• 11. Typically we prepare a mix of plasmid DNA per well as follows: reporter plasmid (100 ng for multiple copies of individual LXREs or 300 ng for promoter fragments containing an LXRE), LXR expression vector (50 ng), and β-galactosidase reporter (100 ng). For these amounts, we normally have the reporters at concentrations of 100 ng/μL and expression vectors at 50 ng/μL. In general, the maximum amount transfected is 500 ng of plasmid DNA per well.

• 12. To reduce variability within (triplicate) samples that have identical DNA mixes, we usually prepare master mixes for 7 wells, which include wells activated with vehicle or ligand (3 each) and an additional well to allow for pipetting errors.

• 13. This order is important for the optimal formation of polymer-DNA complexes.

• 14. We typically use GW3965 at a final concentration of 1 μM from a 10 mM stock diluted in DMSO. Other agonists may need to be prepared at different concentrations and with other solvents. Please refer to the literature available. Those wells that are not activated by LXR agonists should be incubated in media containing the vehicle compound (in this case DMSO) in which the agonist has been dissolved.

• 15. If a high number of plates are being processed, plates can be inverted over a sink to discard medium or PBS, followed by aspiration with a pipette to remove the last drops before addition of PBS or lysis buffer depending on the step.

• 16. Cells may or may not detach and form a pellet. Regardless of whether they detach or not cell lysis still occurs. When pipetting for analysis ensure that the cell pellet is not taken which will affect the reading.

• 17. 5–10 μL of lysate may be enough depending on the strength of signal/ activity, transfection efficiency, and sensitivity of the luminometer .

• 18. This is a starting point. May be reduced depending on sensitivity of the luminometer employed.

• 19. Time may be extended if found to be insufficient to generate a reading.