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Approaches to Recombinant Histamine H3/H4 Receptor Expression in Mammalian Cells

  • Tadeusz Karcz
  • Urszula Cichoń
  • Katarzyna Kieć-KononowiczEmail author
Protocol
Part of the Methods in Pharmacology and Toxicology book series (MIPT)

Abstract

Recombinant receptor techniques are very commonly utilized in pharmacological studies nowadays. This state of affairs is a result of recent development of genetic engineering and DNA cloning methods, which made the expression of foreign genes in various cellular environments possible. The starting point for the application of recombinant proteins in studies on seven-transmembrane receptors (7TMRs) was marked by cloning of the first two 7TMRs-cholinergic and adrenergic receptors in the late 1980s. Resolving of gene sequences for successive receptors enabled the use of expression techniques for already well-characterized 7TMRs, but also for orphan receptors. Within the toolbox of available systems for expression of recombinant receptors, mammalian cells constitute the most frequently used model, since they offer a high probability for maintenance of full functional activity of artificially overexpressed receptors. Taking into consideration the importance of mammalian expression systems in relation to current challenges of molecular pharmacology, some of the available protocols for recombinant 7TMRs production in mammalian cells will be discussed in this chapter.

In the first part, details of transient transfection of CHO cells with genetic construct encoding for fusion protein of histamine H3 receptor and fluorescent protein-mCherry using polyethylenimine as transfection reagent will be given. Then, methods for establishing a monoclonal cell lines stably expressing receptor protein following the lipofection procedure will be presented. Finally, the utility of retroviral expression systems for recombinant receptor production will be discussed using the histamine H4 receptor as an example.

Key words

Histamine H3 and H4 receptors Mammalian expression systems Polyethylenimine (PEI) transfection Recombinant receptors Retroviral transduction Stable transfection 

Notes

Acknowledgments

The authors acknowledge the financial support of Polish National Science Centre within the frame of Meastro grant No. 2011/02/A/NZ4/00031 and Preludium grant No. 2011/01/N/NZ4/01126, which enabled the development and optimization of protocols described in this manuscript.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Tadeusz Karcz
    • 1
  • Urszula Cichoń
    • 1
  • Katarzyna Kieć-Kononowicz
    • 1
    Email author
  1. 1.Department of Technology and Biotechnology of Drugs, Faculty of PharmacyJagiellonian University Medical CollegeKrakówPoland

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