Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Summary

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.

Abstract

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.

Introduction

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.

Protocol

1. Plasmid DNA Purification Extract plasmid DNA using a maxiprep kit according to the manufacturer’s protocol. Resuspend plasmid DNA in 500 µl of TE buffer. Perform a phenol:chloroform:isoamyl alcohol extraction and isopropanol precipitation to remove residual bacterial contaminants. The presence of LPS interferes with transfection9. 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…

Representative Results

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…

Discussion

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 …

Materials

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