Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Nonviral Transfer Technology
  • Published:

Haemoglobin interferes with the ex vivo luciferase luminescence assay: consequence for detection of luciferase reporter gene expression in vivo

Gene Therapy volume 7, pages 1333–1336 (2000)Cite this article

Abstract

The luciferase reporter gene is a useful tool for determining the efficacy of transfection of plasmid DNA and adenovirus-mediated gene transfer in vivo. However, we report here that the haemoglobin present in tissue samples can mask the detection of the luciferase activity and lead to underestimation of the luciferase levels. We evaluated the degree of interference in different organ samples of mice and investigated the possibilities for removal of haemoglobin from tissue samples by: (1) perfusion of the whole animal; (2) different hypotonic treatments lysing preferentially red blood cells; and (3) chromatographic separation. Removal of haemoglobin resulted in significantly improved detection of luciferase activity from tissue samples. The results indicate that the luciferase activity determined in tissue samples may not reflect the actual level of reporter gene expression, if contaminating blood is not taken into consideration.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  1. Thierry AR et al. Systemic gene therapy: biodistribution and long-term expression of a transgene in mice Proc Natl Acad Sci USA 1995 92: 9742–9746

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Fortunati E et al. In vitro and in vivo gene transfer to pulmonary cells mediated by cationic liposomes Biochim Biophys Acta 1996 1306: 55–62

    Article  PubMed  Google Scholar 

  3. Hara T, Tan Y, Huang L . In vivo gene delivery to the liver using reconstituted chylomicron remnants as a novel nonviral vector Proc Natl Acad Sci USA 1997 94: 14547–14552

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Li S, Huang L . In vivo gene transfer via intravenous administration of cationic lipid-protamine-DNA (LPD) complexes Gene Therapy 1997 4: 891–900

    Article  CAS  PubMed  Google Scholar 

  5. Liu F, Qi H, Huang L, Lui D . Factors controlling the efficiency of cationic lipid-mediated transfection in vivo via intravenous administration Gene Therapy 1997 4: 517–523

    Article  CAS  PubMed  Google Scholar 

  6. Barron LG et al. Effects of complement depletion on the pharmacokinetics and gene delivery mediated by cationic lipid-DNA complexes Hum Gene Ther 1998 9: 315–323

    Article  CAS  PubMed  Google Scholar 

  7. Goula D et al. Polyethylenimine-based intravenous delivery of transgenes to mouse lung Gene Therapy 1998 5: 1291–1295

    Article  CAS  PubMed  Google Scholar 

  8. Huard J et al. The route of administration is a major determinant of the transduction efficiency of rat tissues by adenoviral recombinants Gene Therapy 1995 2: 107–115

    CAS  PubMed  Google Scholar 

  9. Yei S et al. Adenovirus gene transfer for cystic fibrosis: quantitative evaluation of repeated in vivo vector administration to the lung Gene Therapy 1994 1: 192–200

    CAS  PubMed  Google Scholar 

  10. Colin M et al. Liposomes enhance delivery and expression of an RGD-oligolysine gene transfer vector in human tracheal cells Gene Therapy 1998 5: 1488–1498

    Article  CAS  PubMed  Google Scholar 

  11. Colin M et al. Cell delivery, intracellular trafficking and expression of an integrin-mediated gene transfer vector in cultured tracheal epithelial cells Gene Therapy 2000 7: 139–152

    Article  CAS  PubMed  Google Scholar 

  12. Harbottle RP et al. An Arg-Gly-Asp/oligolysine peptide: a prototype construct for integrin mediated gene delivery Hum Gene Ther 1998 9: 1037–1047

    Article  CAS  PubMed  Google Scholar 

  13. Wood KV, Lam YA, McElroy WD . Introduction to beetle luciferase and their applications J Biolum Chemilum 1989 4: 289–301

    Article  CAS  Google Scholar 

  14. Leenen PJ et al. Heterogeneity of mouse spleen dendritic cells: in vivo phagocytic activity, expression of macrophage markers, and subpopulation turnover J Immunol 1998 160: 2166–2173

    CAS  PubMed  Google Scholar 

  15. Scher W, Holland JG, Friend C . Hemoglobin synthesis in murine virus-induced leukemic cells in vitro. I. Partial purification and identification of hemoglobins Blood 1971 37: 428–437

    CAS  PubMed  Google Scholar 

  16. Bradford M . A rapid and sensitive method for the quantification of microgram quantities of protein utilizing protein-dye binding Anal Biochem 1976 72: 248–254

    Article  CAS  PubMed  Google Scholar 

  17. Kajiyama N, Nakano E . Isolation and characterization of mutants of firefly luciferase which produce different colors of light Protein Eng 1991 6: 691–693

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the ‘Institut National de la Santé et de la Recherche Médicale’ (INSERM) and from the ‘Association Française de Lutte contre la Mucoviscidose’. We thank Dr A Pavirani of Transgene SA (Strasbourg, France) and Dr M Themis (Imperial College, London) for providing AdMLPLuc and Dr D Gruenert (Gene Therapy Core Center, USA) for the 56FHTE8o− cell line. CC is funded by the Muller Bequest/CF-Trust, the MRC and the March of Dimes Birth Defects Foundation. HS was supported by the Deutsche Forschungsgemeinschaft.

Author information

Author notes

  1. H Schneider

    Present address: University of Erlangen-Nürnberg, Children's Hospital, Loschgestrasse 15, Erlangen, 91054, Germany

  2. M Colin and S Moritz: The first two authors contributed equally to this study

Authors and Affiliations

  1. INSERM U402, Faculté de Médecine Saint-Antoine, Paris, France

    M Colin, S Moritz, J Capeau & MC Brahimi-Horn

  2. Division of Biomedical Sciences, Section of Molecular Genetics, Imperial College, School of Medicine, Sir Alexander Fleming Building, London, UK

    H Schneider & C Coutelle

Authors
  1. M Colin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S Moritz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. H Schneider
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J Capeau
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. C Coutelle
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. MC Brahimi-Horn
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Colin, M., Moritz, S., Schneider, H. et al. Haemoglobin interferes with the ex vivo luciferase luminescence assay: consequence for detection of luciferase reporter gene expression in vivo. Gene Ther 7, 1333–1336 (2000). https://doi.org/10.1038/sj.gt.3301248

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.gt.3301248

Keywords

This article is cited by

Search

Advanced search

Quick links