Ectopic Gene Expression in Macrophages Using in vitro Transcribed mRNA
使用体外转录mRNA在巨噬细胞中进行基因异位表达
发布: 2018年05月20日第8卷第10期 DOI: 10.21769/BioProtoc.2857 浏览次数: 7556
评审: Alka MehraGal HaimovichRan Chen
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参见作者原研究论文
The authors used this protocol in:
Oct 2017
Abstract
Macrophages are immune cells that contribute to host defense through various mechanisms including phagocytosis and antigen presentation. Their antimicrobial capacity is subverted by clinically important intracellular pathogens such as Mycobacterium tuberculosis. The study of host-pathogen interactions using these cells is therefore of considerable interest. Such studies often seek to express tagged proteins to characterize their activities, localizations, and protein-protein interactions. Here, we describe a robust method for transient protein expression in macrophages using mRNA lipoplex transfections.
Keywords: mRNA transfection (mRNA转染)
Macrophage (巨噬细胞)
Protein expression (蛋白质表达)
Background
Typical methods to achieve protein expression, including transfecting DNA using cationic polymers, nucleofection, and viral transduction (Zhang et al., 2009), are particularly difficult in macrophages, as these cells have a potent immune response to various danger signals such as cytosolic DNA. Therefore, conventional methods for exogenous gene delivery result in poor transfection efficiency and cell death. We reasoned that transfecting mRNA, instead of DNA, would be a better alternative to achieve protein expression in macrophages, as suggested by recent reports (Van De Parre et al., 2004; McLenachan et al., 2013). We were able to achieve high transfection efficiencies without loss of macrophage viability (Koster et al., 2017). Our method does not require expensive equipment and can be adapted for expressing exogenous and endogenous proteins.
Materials and Reagents
- μ-Plate 96 Well (ibidi, catalog number: 89626 )
- Sterile RNase-free microfuge tubes (Thermo Fischer Scientific, catalog number: AM12400 )
- DNase and RNase free sterile pipet tips (e.g., Sorenson Bioscience, catalog number: 10350 )
- Gloves (e.g., powder free nitrile gloves, MICROFLEX, catalog number: XC-310 )
- Macrophages
Macrophage cell types that can be used are cell lines such as RAW 264.7 (ATCC, catalog number: TIB-71 ) and primary macrophages such as C57BL/6 bone marrow-derived macrophages (BMDMs). - Cell culture medium
RAW 294.7 cell lines were maintained in Dulbecco Modified Eagle Medium (DMEM, Thermo Fisher Scientific, GibcoTM, catalog number: 11965 ) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Thermo Fisher Scientific, GibcoTM, catalog number: 10082147 ). - CleanCapTM EGFP mRNA (TriLink BioTechnologies, catalog number: L-7601 )
- TransIT®-mRNA Transfection Kit (Mirus Bio, catalog number: MIR 2250 )
- OptiMEM medium (Thermo Fisher Scientific, GibcoTM, catalog number: 31985062 )
- RNaseZapTM (Sigma-Aldrich, catalog number: R2020 )
- mMESSAGE mMACHINE T7 Ultra Kit (Thermo Fisher Scientific, AmbionTM, catalog number: AM1345 )
- MEGAClearTM Transcription Clean-Up Kit (Thermo Fisher Scientific, AmbionTM, catalog number: AM1908 )
- Molecular biology grade water (Corning, catalog number: 46-000-CI )
- Fetal bovine serum (Thermo Fisher Scientific, GibcoTM, catalog number: 16140071 )
- Penicillin and streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
- BglII restriction endonuclease (New England Biolabs, catalog number: R0144S )
- NEBuffer 3.1 (New England Biolabs, catalog number: B7203S )
- Antarctic Phosphatase Reaction Buffer (New England Biolabs, catalog number: B0289S )
- Antarctic Phosphatase (New England Biolabs, catalog number: M0289S )
- QIAquick PCR Purification Kit (QIAGEN, catalog number: 28106 )
- GatewayTM pcDNATM-DEST47 vector (Thermo Fischer Scientific, catalog number: 12281010 )
- Agarose (LE Agarose, GeneMate, catalog number: E-3120-500 )
- 10x MOPS buffer (Fischer Scientific, catalog number: BP2900500 )
- Ethidium bromide (Sigma-Aldrich, catalog number: E1510 )
- RNA Millennium Markers (Thermo Fisher Scientific, AmbionTM, catalog number: AM7150 )
- RNA gel loading dye (Thermo Fischer Scientific, catalog number: R0641 )
- Formaldehyde solution (37%, Sigma-Aldrich, catalog number: 252549 )
- Plasmid linearization reaction mix (see Recipe 1)
- EGFP mRNA transfection reaction mix (see Recipe 3)
- Poly(A) tailing reaction mix (see Recipe 4)
- RNA denaturing gel electrophoresis (see Recipe 5)
- mRNA in vitro transcription reaction mix (see Recipe 6)
- DMEM complete medium (see Recipe 7)
Equipment
- P20 Pipetman (Gilson, catalog number: F123600 )
- P200 Pipetman (Gilson, catalog number: F123601 )
- P1000 Pipetman (Gilson, catalog number: F123602 )
- Multi-channel pipette (Eppendorf, catalog number: 3125000044 )
- Tissue culture CO2 incubator
- Tissue culture hood
- Water bath
- -20 °C freezer
- Epifluorescence microscope (e.g., Nikon, model: Eclipse Ti-E , equipped with 60x; Plan-Apochromat, NA 1.4 oil immersion objective, Ti Z drive, high-resolution monochrome charge-coupled device (CCD) digital camera; Photometric Cool SNAP HQ2 and appropriate filter sets for DAPI, FITC and TexasRed channel)
- Eppendorf Thermomixer® C (Eppendorf, model: ThermoMixer® C , catalog number: 5382000023)
- NanoDrop Spectrophotometer
- Agarose gel electrophoresis system
Software
- Nikon Imaging Software-Elements Advanced Research (NIS-Elements) version 4.40
Procedure
文章信息
版权信息
© 2018 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Chandra, P. and Philips, J. A. (2018). Ectopic Gene Expression in Macrophages Using in vitro Transcribed mRNA. Bio-protocol 8(10): e2857. DOI: 10.21769/BioProtoc.2857.
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分类
分子生物学 > RNA > mRNA 转译
分子生物学 > RNA > 转染
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