Transient Transfection and Reporter Assays to Study Enhancer Activity

Transcriptional activity can be tightly regulated not only by promoter proximal elements, but also by genomic sites far from the gene transcriptional start site. These distal regulatory regions, called enhancers, often house many transcription factor binding sites, and are often sites of active transcription. Enhancers were initially discovered in viral genomes as regions of DNA that enhance transcription independent of orientation or distance to a reporter transcription start site. Today, enhancers are more commonly identified bioinformatically as gene-distal regions that contain high H3K4me1 and/or H3K27ac levels.

We identified a class of putative enhancers bioinformatically, using data that reveals extragenic transcription start sites at base-pair resolution. These sites correlate with several enhancer-like features such as H3K4me1, H3K27ac, DNase hypersensitivity, and cohesin. While these sites correlate with enhancer-like features, the classic assay to identify an enhancer is a luciferase reporter assay. This assay, similar to the first assay that characterized enhancers, uses the luminescene from Firefly luciferase as a readout of transcriptional activity. This gene is placed downstream of a core promoter sequence, and putative enhancers are added distal to the promoter to assay effects of transcriptional activity.

You will be performing transient transfection using the Effectene transfection reagent, and the Promega Dual Luciferase Reporter assay to measure enhancer activity through luminescence. The Dual Luciferase Reporter uses two luciferase constructs: the Firefly luciferase (pFL vectors) as a readout of transcriptional activity, and Renilla luciferase (pRL vector) as a transfection control. Use of the Renilla control not only allows the user to account for differences in transfection efficiency between samples, but also can help account for any differences in cell counts through the assay. The Dual Luciferase Reporter Assay uses two reagents, Buffer II, which contains a substrate reagent for the Firefly luciferase, and the Stop & Glo Buffer, which contains a substrate for the Renilla luciferase as well as a quenching reagent to stop luminescence from the Firefly luciferase. Thus, we will read the Firefly luciferase first, followed by the Renilla luciferase.

In our assay, we will query the enhancer activity of two putative enhancers. We will also perform several controls, including a no transfection control, a GFP transfection control (to determine approximate transfection efficiency), and a background Firefly luciferase activity control (Firefly luciferase construct without any upstream DNA added). Duplicate readings will be taken from each transfection performed.

Outline:
A. Culture S2 cells
B. Transient Transfection with reporter plasmids
C. Dual Luciferase Reporter Assay
D. Appendix: Reagents & Solution

A. Culture S2 cells

  1. Obtain a flask of Drosophila S2 cells from the instructor / TA.
  2. In a hood, resuspend cells by knocking flask hard against the palm of your hand. Remove an aliquot and perform a cell count.
    These cells are semi-adherent cells that grow as single cells. Thus, you will see some cells at the bottom surface of the flask as well as several cells suspended in the media.
  3. Dilute cells in Schneider’s media + 10% serum to 1×10^6 cells/mL in 11 mL.
  4. Aliquot 2 mL (2×10^6 cells) of diluted cells into 5 wells of a 6 well plate. Let incubate at 26C for 24 h before transfection.

B. Transient Transfection

  1. Look at the cells and make sure they are growing well. Try not to disrupt cells too much while moving plate around so that cells are not dislodged from the bottom surface of the plate.
  2. In the hood, for each well to be transfected, combine 100 ng each plasmid to be transfected with 60 uL EC buffer and 1.6 uL Enhancer.
    (This is enough for transfecting 1 well on a 6 well dish. The plasmids you will be given are diluted to 25 ng/uL)

    You will have 5 samples
    Plasmids transfected:
    – No DNA (no transfection control)
    – pActin5C-EGFP (transfection control)
    – pRL-null + pFL empty
    – pRL-null + pFL 01 (enhancer 1)
    – pRL-null + pFL 02 (enhancer 2)

  3. Vortex mixture 1 sec und incubate 2-5 min at RT, then pulse centrifuge.
  4. For each 100 ng DNA in transfection mixture, add 2.5 uL Effectene, vortex 10 sec and incubate on the bench 10 min.
    (For wells with 2 plasmids (total of 200 ng DNA), be sure to add 5 uL Effectene)
  5. While Effectene-DNA incubates, gently aspirate media from cells, rinse with 2 mL of PBS by pipetting PBS to the side of the well and aspirating, then add 1.6 mL Schneider’s media + 10% serum.
    Because S2 cells do not adhere strongly to the bottom of the wells, you will lose cells in this step. To minimize this, be careful not to disrupt cells by not pipetting PBS directly onto cells or shaking the plate.
  6. Label the wells with the DNA you will be adding for transfection.
  7. After DNA-Effectene has incubated 10 min, add 0.4 mL Schneider’s media + 10% serum to each DNA-Effectene mix, then pipette up & down twice to mix.
  8. Dispense 0.4 mL appropriate transfection mix into each well and swirl the dish to mix.
  9. Incubate 24 h at 26C before cell lysis and luciferase analysis.
    Subsequent expression assay can be performed anytime from 24-72 h later. The ideal time may need to be determined empirically for any given transfection experiment.

C. Dual Luciferase Reporter Assay

  1. We will be using the dual luciferase reporter assay system marketed by Promega (E1910). The TA will combine the Luciferase Assay Buffer II and substrate, the “Stop & Glo” buffer and substrate, and will dilute 5X Passive Lysis Buffer from this kit to 1X with ddH2O. If performing yourself, store both reagents according to the manufacturer’s instruction.
  2. Briefly look at cells under the microscope to confirm that there is no contamination. Look for GFP under the microscope to assay transfection efficiency.
    (S2 cells are difficult to transfect and we typically only see about 10% GFP+ cells using this protocol. Again, be careful not to disrupt cells so you do not dislodge from the plate bottom.)
  3. Lyse cells by aspirating media from cells and adding 300 uL 1X Passive Lysis Buffer per well. Place on rocker for 15 min to lyse cells.
    (The GFP vector was just a transfection control, you do not need it for luciferase analysis.)
  4. Following the lysis, shake the plate gently to mix cell lysates without mixing wells. Aliquot 20 uL of each sample to provided tubes. Try to avoid getting the sample on the sides of the tube by pipetting directly at the bottom of the tube. We will be performing luminescence reading in duplicate, so you should have 10 tubes at this point (5 samples, duplicate readings). Keep samples ion ice following 15 min lysis.
  5. Bring samples to luminometer and TA to continue the assay.
  6. With TA, read background luminescence.
    We recommend 10 sec readings.
  7. Add 100 uL Buffer II, mix by pipetting up and down twice and reads luminescence immediately.
    Do not vortex here to mix and avoid getting excess buffer or sample on the sides of the tube.
    Luciferase activity decreases with time, so be sure to read luminescence right after adding the reagent.
  8. Remove the tube from the luminometer, add 100 uL “Stop & Glo” buffer, mix by vortexing and read luminescence.

D. Appendix: Reagents and Solutions

Culture Medium
Schneider’s Media + 1% serum

1X PBS

6 well plate for each group (x8)
Falcon Tissue Culture Plate (No: 353046; Fisher Cat No. 08-772-1B)

Effectene (x1)
Effectene Transfection Reagent (Qiagen, Cat. No. 301425)

Reporter Assay System (x1)
Dual-Luciferase Reporter Assay (Promega, Cat. No. E1910)