Pharmacological Applications of Baculovirus‐Mediated Protein Expression in Mammalian Cells

J. Patrick Condreay1, Christian A. Watson1

1 GlaxoSmithKline Molecular Discovery Research, Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 6.11
DOI:  10.1002/0471141755.ph0611s49
Online Posting Date:  June, 2010
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Abstract

The development of cell‐based assays for cellular receptors, ion channels, and transporters requires the delivery and expression of transgenes. Viral‐mediated gene delivery is a particularly attractive approach for this purpose because of its efficiency and potential to deliver genes to a wide variety of cell types. Recombinant baculoviruses, long used to deliver genes to insect cells for overexpression, also effectively transfer genes to mammalian cells. The only required modification to the virus for this purpose is the addition of transgene expression cassettes controlled by mammalian cell‐active promoters. These so‐called BacMam viruses are useful for developing mammalian cell‐based assays for investigating the function of recombinant proteins and for assessing the action of pharmacological modulators of their function. The use of such viruses is gaining popularity because of the ease of optimizing assay conditions, the ability to deliver multiple gene products, and of their flexibility in terms of host cells and levels of transgene expression. BacMam‐mediated assays may be used for studying a wide variety of target proteins and assay technologies. Described in this unit is an example of BacMam‐mediated gene delivery to configure a cell‐based assay for pharmacological assessment of a G protein‐coupled receptor. A protocol is also provided describing the use of a GFP‐expressing BacMam to assess the susceptibility of new cell lines to transduction by the virus. Curr. Protoc. Pharmacol. 49:6.11.1‐6.11.12. © 2010 by John Wiley & Sons, Inc.

Keywords: BacMam gene delivery; cell‐based assays; transient expression

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: BacMam Virus‐Mediated Transduction of a G Protein–Coupled Receptor into Mammalian Cells
  • Basic Protocol 2: Assessment of GPCR Receptor Functional Expression Using a FLIPR Calcium Mobilization Assay
  • Support Protocol 1: Assessing Susceptibility of Cell Lines to BacMam Transduction Using a BacMam‐GFP Virus
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: BacMam Virus‐Mediated Transduction of a G Protein–Coupled Receptor into Mammalian Cells

  Materials
  • Cells (e.g., U‐2 OS) growing in tissue culture (ATCC)
  • Dulbecco's phosphate‐buffered saline (D‐PBS; Invitrogen)
  • Cell dissociation buffer, enzyme‐free, PBS‐based (Invitrogen, cat. no. 13151‐014)
  • Complete growth medium (see recipe)
  • Receptor BacMam virus (see recipe)
  • G‐protein BacMam virus (see recipe)
  • 10‐ml plastic pipets
  • Hemacytometer
  • 50‐ml conical tubes
  • 384‐well clear bottom plates, tissue culture treated (Greiner Bioscience, cat. no. 781091)
NOTE: Cell culture procedures are always performed using sterile technique, with incubations in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 2: Assessment of GPCR Receptor Functional Expression Using a FLIPR Calcium Mobilization Assay

  Materials
  • Transduced cells in 384‐well tissue culture‐treated plates (see protocol 1)
  • Brilliant black/Fluo‐4 loading buffer (see recipe)
  • Ligand dilution buffer (see recipe)
  • FLIPR Tetra Fluorometric Imaging Plate Reader (Molecular Devices)
  • Computer with Excel
  • Curve‐fitting program (e.g., GraphPad Prism)

Support Protocol 1: Assessing Susceptibility of Cell Lines to BacMam Transduction Using a BacMam‐GFP Virus

  Materials
  • Cells plated in multi‐well dish at 50% to 70% confluence
  • Sodium butyrate (Sigma)
  • BacMam GFP virus (see recipe)
  • Complete growth medium (see recipe)
  • 0.2‐µm filters
  • 15‐ml tubes, sterile
  • Any inverted microscope with fluorescence excitation and detection capability such as the Leica DMI 4000 microscope
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Figures

Videos

Literature Cited

Literature Cited
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