Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Podsumowanie

Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell’s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells.

Streszczenie

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been described, certain types of cells are resistant to many of these methods and yield low transfection efficiency¹, potentially hindering research in those cell types. In this protocol, we present an optimized transfection procedure to introduce luciferase reporter genes as a plasmid DNA into the RAW264.7 macrophage cell line. Two different types of transfection reagents (lipid-based and polyamine-based) are described, and important notes are given throughout the protocol to ensure that the RAW264.7 cells are minimally altered by the transfection procedure and any experimental data obtained are the direct results of the experimental treatment. While transfection efficiency may not be higher compared to other transfection methods, the described procedure is robust enough for detecting luciferase signal in RAW264.7 without changing the physiological response of the cells to stimuli.

Wprowadzenie

Transfection of nucleic acids in cells has a diverse application in scientific research. Examples include (1) reporter genes to study the role of different gene elements in gene expression, (2) protein expression plasmids to overexpress the protein of interest, and (3) small interfering RNA to downregulate gene expression. By manipulating the expression level of particular genes and measuring the differential effect of such manipulations, researchers can deduce the gene functions in the chosen biological systems. Not all transfection methods provide the same transfection efficiencies, and even the same transfection method does not transfect all cell types equally¹. Hence, different transfection methods have been developed such as calcium phosphate method, DEAE dextran, cationic lipid transfection, cationic non-lipid polymer transfection, electroporation, and nucleofection^2,3.

Transfection into macrophages is especially difficult due to the fact that macrophages are professional phagocytes that are very sensitive to foreign materials including bacteria derived (methylated) DNA⁴. Introduction of foreign DNA activates the Toll-like receptor 9 (TLR9) pathway leading to the production of cytokines and nitric oxide^5,6. These activated macrophages may then be less responsive to treatment that the researchers intend to examine.

Our lab routinely transfects the RAW264.7 macrophage cell line with luciferase reporter genes, and we have developed a protocol that is robust enough to have luciferase signal significantly higher than background, but also gentle enough for macrophages to remain at their resting state. The behaviours of the transfected cells were evaluated by a firefly-luciferase reporter gene harbouring the promoter region of IκBζ (pGL3- IκBζ). IκBζ expression is upregulated by the bacterial cell wall component lipopolyssacharide (LPS)^7,8, and downregulated by the anti-inflammatory cytokine, Interleukin-10 (IL-10)⁸. To account for transfection variation among wells, we co-transfect a control plasmid containing the Renilla luciferase gene (e.g., phRL-TK) for normalization purposes. The protocol described is optimized after testing various parameters including timing of transfection, type of transfection reagents, amounts of transfection reagents and of plasmid DNA, as well as ratio of transfection reagent to plasmid DNA. The two transfection reagents included in this protocol are (1) a lipid-based transfection reagent and (2) a protein/polyamine-based transfection reagent.

Protokół

1. Plasmid DNA Purification

1. Extract plasmid DNA using a maxiprep kit according to the manufacturer’s protocol. Resuspend plasmid DNA in 500 µl of TE buffer.
2. Perform a phenol:chloroform:isoamyl alcohol extraction and isopropanol precipitation to remove residual bacterial contaminants. The presence of LPS interferes with transfection⁹.
   1. Add 500 µl of phenol:chloroform:isoamyl alcohol (25:24:1, pH 8) to the plasmid DNA and shake vigorously for 15 sec. Phenol causes severe skin burns and is an anesthetic so burns are not always felt until there is severe damage. Perform phenol:chloroform:isoamyl extraction in the fume hood and use caution.
3. Incubate the mixture for 5 min at RT. Centrifuge at 13,000 x g for 10 min at RT. Transfer 350 µl of upper aqueous phase into a new 1.5 ml collection tube.
4. Add 350 µl of TE buffer to the tube that contains the lower organic phase. Shake vigorously for 15 sec. Centrifuge at 13,000 x g for 5 min at RT. Transfer 350 µl of upper aqueous phase to the same 1.5 ml collection tube.
5. Add 70 µl of 3 M sodium acetate (pH 5.2) and mix well. Add 700 µl of 100% isopropanol. Mix well and incubate 10 min at RT. Centrifuge at 13,000 x g for 10 minat 4 °C. Remove the supernatant.
6. Add 500 µl of 75% ethanol to the pellet. Vortex samples to mix and centrifuge at 5,000 x g for 5 min at 4 °C. Remove the supernatant. DNA is in the pellet.
7. Open the cap of the tube and air dry the pellet at RT for 5 - 10 min. The pellet should become translucent. Add 500 µl of TE buffer to resuspend the DNA pellet. Heat at 50 - 60 °C for 10 min to dissolve the DNA pellet.
8. Measure the absorbance at 260 nm (A260) and 280 nm (A280) with a spectrometer. Calculate the concentration of DNA by multiplying the A260 value by 50 ng/µl. Assess the quality of DNA by calculating the A260/A280 ratio. DNA with high quality should give an A260/A280 ratio of 1.8 - 2.0.

2. Cell Culturing and Seeding

1. Culture RAW264.7 cells in DMEM supplemented with 9% fetal calf serum (DMEM/9% FCS) in a 37 °C, 5% CO₂ incubator. During each passage, detach the cells from the plate by continuous pipetting using a Pasteur pipette. Passage the cells every 2 days, and seed 1.5 - 2 million cells on a 10 cm tissue culture treated dish as the stock. Thaw a new stock of cells every 5 - 6 weeks.
2. On the day of transfection, seed 200,000 cells per well in a 24-well plate in a volume of 500 µl of DMEM/9% FCS. Incubate the cells for 4 hr in the 37 °C incubator.
3. Transfect cells either with the lipid-based reagent (step 3.1) or the protein/polyamine-based reagent (step 3.2).

3. Transfection

1. Lipid-based transfection:
   1. Warm up the transfection reagent to RT in the dark. Warm up serum-free medium and DMEM/9% FCS to 37 °C.
   2. Add 0.5 µg of plasmid DNA to 50 µl of serum-free medium in a 1.5 ml tube. Add 2 µl of transfection reagent with the pipette tip below the surface of the liquid. Vortex for 6 sec.
   3. Leave the reagent/DNA mixture in the dark at RT for 30 min.
   4. During the 30 min incubation, remove the media from the well and add 250 µl of fresh DMEM/9% FCS to each well.
   5. After 30 min, add 250 µl of DMEM/9% FCS to the reagent/DNA mixture. Mix well by pipetting up and down.
   6. Add 300 µl of the diluted mixture to each well. Incubate for 2 - 4 hr in a 37 °C, 5% CO₂ incubator.
   7. Remove transfection solution and add 500 µl of DMEM/9% FCS. Incubate for 24 - 48 hr in a 37 °C, 5% CO₂ incubator before cell stimulation.
2. Protein/polyamine-based transfection:
   1. Warm up serum-free medium and DMEM/9% FCS to 37 °C.
   2. Add 0.75 µl of transfection reagent to 18.75 µl of serum-free medium. Vortex briefly to mix. Incubate at RT for 5 min. Add 0.5 µl of 1 µg/µl DNA into the diluted transfected reagent. Incubate at RT for 10 min.
   3. During the 10 min incubation, remove media from each well of the 24-well plate and add 250 µl fresh DMEM/9% FCS to each well.
   4. After 10 min, add 250 µl of DMEM/9% FCS into the reagent/DNA mixture.
   5. Add 270 µl of the diluted mixture to the well. Incubate in a 37 °C and 5% CO₂ incubator for 2 - 4 hr.
   6. Remove transfection solution and add 500 µl of DMEM/9% FCS. Incubate for 24 - 48 hr in a 37 °C, 5% CO₂ incubator.

4. Cell Stimulation and Luciferase Assay

1. Replace media in the well with 250 µl of fresh DMEM/9% FCS.
2. Add 50 µl of LPS or LPS+IL-10 at the desired concentrations to the well. Return the plate to the 37 °C, 5% CO₂ incubator. Stimulate for 2 - 6 hr.
3. Remove stimulation solution by aspiration, wash with ice cold PBS, and add 200 µl of 1x Passive Lysis Buffer. Rock at RT for 30 min. Scrape and transfer the lysate to 1.5 ml tubes and remove cell debris by centrifuging at 20,000 x g for 10 min at 4 °C.
4. Transfer 40 µl of cleared lysate (supernatant) to a white, clear bottom 96-well plate for the determination of luciferase signals according to the manufacturer’s protocol.
5. Read the luciferase signal with a luminometer across the entire visible light spectrum.

5. Data Analysis

1. Calculate the firefly:Renilla ratio by dividing individual values of firefly luciferase by the values of Renilla luciferase from each well.
2. Calculate the fold change by dividing the firefly:Renilla ratio of the treated well by that of the untreated well.

Wyniki

Figure 1 compares the transfection efficiency of the two transfection reagents in RAW264.7. The lipid-based reagent typically gave about 25% transfection rate while the protein/polyamine-based transfection resulted in about 5% efficiency (Figure 1A). The difference in transfection efficiency was also observed in luciferase signals in RAW264.7 cells transfected with the pGL3-IκBζ promoter reporter (Figure 1B). Addition of LPS to these transfected cells increased...

Dyskusje

The protocol described here does not solely focus on transfection efficiency, but aims to strike a balance between efficiency and preservation of the physiological states of the cells. Specifically, our procedure succeeds in minimizing the toxicity of transfection reagent and maximizing luciferase signal.

One critical step in the protocol is the health of the cells. Overgrown cultures are not suitable for transfection as their physiology changes, and continuous culturing of RAW264.7 cells for ...

Materiały

Name	Company	Catalog Number	Comments
PureLink HiPure Plasmid Maxiprep Kit	Life Technologies	K210007	Any maxiprep kit will work
Phenol:chloroform:isoamyl alcohol	Life Technologies	15593-049	Molecular Biology Grade. Phenol is toxic so work in the fume hood, if possible. Use the lower clear organic layer if two layers of liquid form in the container.
DMEM	Thermo Scientific	SH30243.01	Warm in 37°C water bath before use.
Fetal Bovine Serum	Thermo Scientific	SH30396.03	Inactivated at 56°C water bath for 45 minutes before use.
Opti-MEM	Life Technologies	31985-070	Warm to at least room temperature before use.
XtremeGene HP DBA transfection reagent	Roche	6366236001	Warm to room temperature before use.
GeneJuice	EMD Millipore	70967	Warm to room temperature before use.
5X Passive Lysis Buffer	Promega	E1941	30 ml is included in the Dual Luciferase Reporter Assay System
Dual Luciferase Reporter Assay System	Promega	E1910