Skip to main content
SpringerLink
Log in

Promoter Assay Using Luciferase Reporter Gene in the A549 Cell Line

  • Protocol
  • First Online:
Molecular Genetics of Asthma

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1434))

Abstract

The development of reporters systems has simplified the study of promoter activity in different areas of knowledge, and represents an easy and fast approach to study genetic variations. In this chapter, we show a transfection protocol of A549 lung epithelial cells with a reporter vector, using the Luciferase-Renilla dual system for studying the variations caused by several polymorphisms in the promoter region of a gene.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Ramos E, Espinosa E, Vega A et al (2005) Expresión de genes reporteros en la línea QT 35 utilizando la transfección con liposomas. Rev Salud Anim 27:152–158

    Google Scholar 

  2. Jiang T, Xing B, Rao J (2008) Recent developments of biological reporter technology for detecting gene expression. Biotechnol Genet Eng Rev 25:41–76

    Article  CAS  PubMed  Google Scholar 

  3. Zhuang LL, Jin R, Zhu LH et al (2013) Promoter characterization and role of cAMP/PKA/CREB in the basal transcription of the mouse ORMDL3. Plos One 8(4):e60630. doi:10.1371/journal.pone.0060630

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Keen JC, Cianferoni A, Florio G et al (2005) Characterization of a novel PMA-inducible pathway of interleukin-13 gene expression in T cells. Inmunology 117:29–37

    Google Scholar 

  5. Binia A, Van Stiphout N, Liang L et al (2013) A polymorphism affecting MYB binding within the promoter of the PDCD4 gene is associated with severe asthma in children. Hum Mutat 34:1131–1139

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Hu M, Ou-Yang HF, Wu CG et al (2014) Notch signaling regulates col1α1 and col1α2 expression in airway fibroblasts. Exp Biol Med 239:1589–1596

    Article  Google Scholar 

  7. Ou-Yang HF, Wu CG, Qu S et al (2013) Notch signaling downregulates MUC5AC expression in airway epithelial cells through Hes1-dependent mechanisms. Respiration 86:341–346

    Article  CAS  PubMed  Google Scholar 

  8. Ni ZH, Tang JH, Cai ZY et al (2011) A new pathway of glucocorticoid action for asthma treatment through the regulation of PTEN expression. Respir Res 12:47

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Matsukura S, Kurokawa M, Homma T et al (2013) Basic research on virus-induced asthma exacerbation: inhibition of inflammatory chemokine expression by fluticasone propionate. Int Arch Allergy Immunol 161(Suppl 2):84–92

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Tanner FC, Carr DP, Nabel GJ et al (1997) Transfection of human endothelial cells. Cardiovasc Res 35:522–528

    Article  CAS  PubMed  Google Scholar 

  11. Castro FO, Portelles Y (1997) Transfección de ADN a células de mamíferos. Biotecnol Apl 14:149–161

    CAS  Google Scholar 

  12. Schenborn E, Groskreutz D (1999) Reporter gene vectors and assays. Mol Biotechnol 13:29–44

    Article  CAS  PubMed  Google Scholar 

  13. Köhler S, Belkin S, Schmid RD (2000) Reporter gene bioassays in environmental analysis. Fresenius J Anal Chem 366:769–779

    Article  PubMed  Google Scholar 

  14. Wet JR, Wood KV, Helinski DR et al (1985) Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli. ProcNatl Acad Sci U S A 82:7870–7873

    Article  Google Scholar 

  15. Dual-lucifersae reporter assay system promega. Technical Manual. Promega Corporation.http://www.promega.com

  16. Hou Y, Cheng B, Zhou M et al (2014) Searching for synergistic bronchodilators and novel therapeutic regimens for chronic lung diseases from a traditional Chinese medicine, Qingfei Xiaoyan Wan. Plos One 9(11), e113104

    Article  PubMed  PubMed Central  Google Scholar 

  17. Haque R, Hakim A, Moodley T et al (2013) Inhaled long-acting β2 agonists enhance glucocorticoid receptor nuclear translocation and efficacy in sputum macrophages in COPD. J Allergy Clin Immunol 132:1166–1173

    Article  CAS  PubMed  Google Scholar 

  18. Harrop CA, Gore RB, Evans CM et al (2013) TGF-β2 decreases baseline and IL-13-stimulated mucin production by primary human bronchial epithelial cells. Exp Lung Res 39:39–47

    Article  CAS  PubMed  Google Scholar 

  19. Park IH, Um JY, Cho JS et al (2014) Histamine promotes the release of interleukin-6 via the H1R/p38 and NF-κB pathways in nasal fibroblasts. Allergy Asthma Immunol Res 6:567–572

    Article  PubMed  PubMed Central  Google Scholar 

  20. Haj-Selem I, Fakhfakh R, Bérubé J et al (2014) MicroRNA-19a enhances proliferation of bronchial epithelial cells by targeting TGFβR2 gene in severe asthma. Allergy. doi:10.1111/all.12551

    Google Scholar 

  21. Garbacki N, Di Valentin E, Huynh-Thu VA et al (2010) MicroRNAs profiling in murine models of acute and chronic asthma: a relationship with mRNAs targets. Plos One 6(1):e16509

    Article  Google Scholar 

  22. Takyar S, Vasavada H, Zhang J et al (2013) VEGF controls lung Th2 inflammation via the miR-1-Mpl (myeloproliferative leukemia virus oncogene)-P-selectin axis. J Exp Med 210:1993–2010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Jude JA, Dileepan M, Kannan MS et al (2012) miR-140-3p regulation of TNF-α-induced CD38 expression in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 303:460–468

    Article  Google Scholar 

  24. 260/280 and 260/230 Ratios. Technical bulletin. Thermo Fisher Scientific.http://www.nanodrop.com/Library/T009-NanoDrop%201000-&-NanoDrop%208000-Nucleic-Acid-Purity-Ratios.pdfAccessed. Accessed 17 Sept 2014

  25. Colosimo A, Goncz KK, Holmes AR et al (2000) Transfer and expression of foreign genes in mammalian cells. Biotechniques 29:314–331

    CAS  PubMed  Google Scholar 

  26. Franco AY, Longart M (2009) Aplicaciones de la proteína verde fluorescente (GFP) en la biología celular y en la visualización del sistema nervioso. Revista de Estudios Transdisciplinarios 1:84–96

    Google Scholar 

  27. Yang T, Sinai PS, Kitts PA et al (1997) Quantification of gene expression with a secreted alkaline phosphatase reporter system. Biotechniques 23:1110–1114

    CAS  PubMed  Google Scholar 

  28. Ghim CM, Lee SK, Takayama S et al (2010) The art of reporter proteins in science: past, present and future applications. BMB Rep 43:451–460

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by grants of the Junta de Castilla y León ref. GRS1047/A/14 and BIO/SA73/15, and by the project “Efecto del Ácido Retinóico en la enfermedad alérgica. Estudio transcripcional y su traslación a la clínica,” PI13/00564, integrated into the “Plan Estatal de I + D + I 2013–2016” and cofunded by the “ISCIII-Subdirección General de Evaluación y Fomento de la investigación” and the European Regional Development Fund (FEDER).

Author information

Authors and Affiliations

  1. Department of Clinical Biochemistry, University Hospital of Salamanca, Paseo de San Vicente 58, Salamanca, 37007, Spain

    Elena Marcos-Vadillo

  2. Salamanca Institute for Biomedical Research (IBSAL), Salamanca, Spain

    Elena Marcos-Vadillo

  3. IBSAL, Salamanca Institute for Biomedical Research, Salamanca, Spain

    Asunción García-Sánchez

  4. Department of Biomedical and Diagnostic Sciences, University of Salamanca, Spain

    Asunción García-Sánchez

Authors
  1. Elena Marcos-Vadillo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Asunción García-Sánchez
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Elena Marcos-Vadillo .

Editor information

Editors and Affiliations

  1. Department of Clinical Biochemistry, University Hospital of Salamanca, Salamanca, Spain

    María Isidoro García

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Marcos-Vadillo, E., García-Sánchez, A. (2016). Promoter Assay Using Luciferase Reporter Gene in the A549 Cell Line. In: Isidoro García, M. (eds) Molecular Genetics of Asthma. Methods in Molecular Biology, vol 1434. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3652-6_14

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3652-6_14

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3650-2

  • Online ISBN: 978-1-4939-3652-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation