Transfektion RAW264.7 Celler med en luciferasereportergen
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
Transfektion af nukleinsyrer i celler har et mangfoldigt program i videnskabelig forskning. Eksempler indbefatter (1) reportergener at undersøge rollen af de forskellige gen-elementer i gen-ekspression, (2) protein ekspressionsplasmider at overudtrykke proteinet af interesse, og (3) små interfererende RNA at nedregulere genekspression. Ved at manipulere ekspressionsniveauet af bestemte gener og måle forskellen virkningen af sådanne manipulationer kan forskerne udlede de genfunktioner i de udvalgte biologiske systemer. Ikke alle transfektionsfremgangsmåder giver samme transfektionseffektiviteter, og selv den samme transfektion metode ikke transficere alle celletyper ligeligt 1. Derfor er der blevet udviklet forskellige transfektionsmetoder, såsom calciumphosphat-metoden, DEAE-dextran, kationisk lipid transfektion, kationisk ikke-lipid polymer transfektion, elektroporering og nucleofection 2,3.
Transfektion ind makrofager er ESPEcielt vanskelig på grund af det faktum, at makrofager er professionelle fagocytter, der er meget følsomme over for fremmede materialer herunder bakterier afledt (methyleret) DNA 4. Introduktion af fremmed DNA aktiverer Toll-like receptor 9 (TLR9), der fører til produktion af cytokiner og nitrogenoxid 5,6. Disse aktiverede makrofager kan så være mindre lydhøre over for behandling, at forskerne har til hensigt at undersøge.
Vores laboratorium rutinemæssigt transfects den RAW264.7 makrofagcellelinie med luciferase reportergener, og vi har udviklet en protokol, der er robust nok til at have luciferase signal betydeligt højere end baggrunden, men også blide nok til makrofager til at forblive på deres hviletilstand. Adfærd af de transficerede celler blev evalueret af en ildflue-luciferase reportergen huser promotorregionen af IκBζ (pGL3- IκBζ). IκBζ udtryk opreguleres af den bakterielle cellevæg komponent læbeopolyssacharide (LPS) 7,8, og nedreguleres af anti-inflammatoriske cytokin, interleukin-10 (IL-10) 8. At tage højde for transfektion variation blandt brønde, vi co-transficere en kontrol plasmid indeholdende Renilla-luciferase-genet (f.eks phRL-TK) til normaliserings- formål. Den beskrevne protokol er optimeret efter afprøvning af forskellige parametre, herunder timingen af transfektion, type transfektionsreagenser, mængderne af transfektionsreagenser og plasmid-DNA, såvel som forholdet mellem transfektionsreagens til plasmid-DNA. De to transfektionsreagenser inkluderet i denne protokol er (1) en lipid-baserede transfektionsreagens og (2) et protein / polyamin-baserede transfektionsreagens.
Protocol
Representative Results
Discussion
Protokollen beskrevet her ikke udelukkende fokusere på transfektion effektivitet, men har til formål at finde en balance mellem effektivitet og bevarelse af de fysiologiske tilstande af cellerne. Specifikt vores procedure lykkes med at minimere toksiciteten af transfektionsreagens og maksimere luciferase signal.
Et afgørende skridt i protokollen er sundhed af cellerne. Tilgroet kulturer er ikke egnede til transfektion som deres fysiologi forandringer, og kontinuerlig dyrkning af RAW…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This study was funded by Canadian Institutes for Health Research (CIHR) grant. STC holds a doctoral research award from the CIHR and the Michael Smith Foundation. EYS holds a CIHR scholarship. The CIHR Transplantation Training Program also provided graduate scholarships to STC, EYS and SS.
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 |
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