Summary

转染RAW264.7细胞用荧光素酶报告基因

Published: June 18, 2015
doi:
Please note that all translations are automatically generated. Click here for the English version.

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Disclosures

The authors have nothing to disclose.

Acknowledgements

Materials

PureLink HiPure Plasmid Maxiprep KitLife TechnologiesK210007Any maxiprep kit will work
Phenol:chloroform:isoamyl alcoholLife Technologies15593-049Molecular 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.
DMEMThermo ScientificSH30243.01Warm in 37 °C water bath before use.
Fetal bovine serumThermo ScientificSH30396.03Inactivated at 56 °C water bath for 45 min before use.
Opti-MEMLife Technologies31985-070Warm to at least room temperature before use.
XtremeGene HP DBA transfection reagentRoche6366236001Warm to room temperature before use.
GeneJuiceEMD Millipore70967Warm to room temperature before use.
5x Passive Lysis BufferPromegaE194130 ml is included in the Dual Luciferase Reporter Assay System
Dual Luciferase Reporter Assay SystemPromegaE1910

References

  1. Maurisse, R., et al. Comparative transfection of DNA into primary and transformed mammalian cells from different lineages. BMC Biotechnol. 10 (1), 9-18 (2010).
  2. Thompson, C. D., Frazier-Jessen, M. R., Rawat, R., Nordan, R. P., Brown, R. T. Evaluation of methods for transient transfection of a murine macrophage cell line RAW 264.7. Biotechniques. 27 (4), 824-835 (1999).
  3. Kim, T., Eberwine, J. Mammalian cell transfection: the present and the future. Analytical and Bioanalytical Chemistry. 397 (8), 3173-3178 (2010).
  4. Stacey, K. J., Sweet, M. J., Hume, D. A. Macrophages ingest and are activated by bacterial DNA. Journal of immunology. 157 (5), 2116-2122 (1996).
  5. Jiang, W., Reich, I. C., Pisetsky, D. S. Mechanisms of activation of the RAW264.7 macrophage cell line by transfected mammalian DNA. Cell Immunol. 229 (1), 31-42 (2004).
  6. Jiang, W., Pisetsky, D. S. The induction of HMGB1 release from RAW 264.7 cells by transfected DNA. Mol Immunol. 45 (7), 2038-2046 (2008).
  7. Hargreaves, D. C., Horng, T., Medzhitov, R. Control of inducible gene expression by signal-dependent transcriptional elongation. Cell. 138 (1), 129-147 (2009).
  8. Cheung, S. T., So, E. Y., Chang, D., Ming-Lum, A., Mui, A. L. Interleukin-10 inhibits lipopolysaccharide induced miR-155 precursor stability and maturation. PLoS One. 8 (8), e71336 (2013).
  9. Weber, M., Moller, K., Welzeck, M., Schorr, J. Short technical reports. Effects of lipopolysaccharide on transfection efficiency in eukaryotic cells. Biotechniques. 19 (6), 930-940 (1995).
  10. Berghaus, L. J., et al. Innate immune responses of primary murine macrophage-lineage cells and RAW 264.7 cells to ligands of Toll-like receptors. Comp Immunol Microbiol Infect Dis. 33 (5), 443-456 (2000).
  11. Fiszer-Kierzkowska, A., et al. Liposome-based DNA carriers may induce cellular stress response and change gene expression pattern in transfected cells. BMC Mol Biol. 12 (1), 27-36 (2011).
  12. Jacobsen, L., Calvin, S., Lobenhofer, E. Transcriptional effects of transfection: the potential for misinterpretation of gene expression data generated from transiently transfected cells. Biotechniques. 47 (7), 617-626 (2009).
Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Play Video

Cite This Article
Cheung, S. T., Shakibakho, S., So, E. Y., Mui, A. L. Transfecting RAW264.7 Cells with a Luciferase Reporter Gene. J. Vis. Exp. (100), e52807, doi:10.3791/52807 (2015).

View Video