Generation of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) Using Directed Molecular Evolution

Ying Pei1, Shuyun Dong1, Bryan L. Roth1

1 University of North Carolina, Chapel Hill, North Carolina
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 4.33
DOI:  10.1002/0471142301.ns0433s50
Online Posting Date:  January, 2010
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Abstract

G protein–coupled receptors (GPCRs) and their signal transductions are important for both physiological and pathological processes in neuron systems. Neuronal GPCRs activated by synthetic ligands have been created by designed mutagenesis for studying their functions and signal pathways. However, these engineered GPCRs have problems, such as their high constitutive activity. To overcome this drawback, a new generation of receptors termed designer receptors exclusively activated by designer drugs (DREADDs), have been designed. DREADDs are exclusively activated by synthetic ligands, but are insensitive to their endogenous ligand and have no constitutive activity, which provides the ability to selectively modulate signal transduction of certain GPCRs in vitro and in vivo. This protocol provides detailed instructions for creating DREADDs using directed molecular evolution. The procedures to generate DREADDS include GPCR functional expression in yeast, mutant GPCR library generation, and high‐throughput yeast screening. These methods are general and suitable for any GPCRs that can be functionally expressed in yeast. Curr. Protoc. Neurosci. 50:4.33.1‐4.33.25. © 2010 by John Wiley & Sons, Inc.

Keywords: DREADD; RASSL; directed molecular evolution; synthetic biology; chemical biology

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Expression and Testing of the Function of the Target GPCR in Yeast
  • Basic Protocol 2: Generating Yeast Mutant Libraries Expressing Randomly Mutated Receptors
  • Basic Protocol 3: Yeast Mutant Library Screening and Liquid Yeast Growth Assays
  • Basic Protocol 4: Determine the Mutation Site(s) and Confirm the Pharmacological Profile of Selected Candidates
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Expression and Testing of the Function of the Target GPCR in Yeast

  Materials
  • Target GPCR
  • Expression vector (e.g., p416GPD)
  • YAPD agar plates (see recipe)
  • Yeast strain (e.g., YB1)
  • YAPD (yeast extract/adenine/peptone/dextrose) medium (see recipe)
  • –URA agar plates: synthetic complete (SC) medium plate lacking uracil (see recipe)
  • Herring sperm DNA (e.g., Promega, cat. no. D181A)
  • 40% (w/v) polyethylene glycol (PEG) 4000 (e.g., Sigma, cat. no. 95904‐1kg‐F) made in 0.1 M lithium acetate (see recipe), filter sterilized
  • 0.1 M lithium acetate in TE buffer, pH 7.5 (see recipe), filter‐sterilized
  • –URA medium: synthetic complete (SC) medium lacking uracil (see recipe)
  • 30% (w/v) glycerol, sterile
  • Liquid growth assay medium (see recipe)
  • Test (drug) compounds
  • 15‐ and 50‐ml tubes, sterile
  • Humidified 25°C and 30°C cell culture incubators
  • Temperature‐adjustable incubator shaker
  • 1.5‐ml microcentrifuge tubes, sterile
  • 42°C water bath
  • 96‐well flat‐bottom plates with lids, low attachment (e.g., Costar, cat. no. 3937), sterile
  • Microplate reader
  • Parafilm
  • Plate shaker
  • Multi‐channel pipettors (8‐channel and 12‐channel) and sterile pipet tips
  • Additional reagents and equipment for performing molecular biology techniques (e.g., see appendix 1A)

Basic Protocol 2: Generating Yeast Mutant Libraries Expressing Randomly Mutated Receptors

  Materials
  • GeneMorph II random mutagenesis kit (Stratagene, cat. no. 200550)
  • Yeast construct expressing the wild‐type target GPCR (sequence and functions confirmed by protocol 1)
  • PCR primers (Fig. )
  • Agarose gel
  • QIAquick gel extraction kit (Qiagen, cat. no. 28704)
  • Restriction enzymes and 10× buffers
  • Calf intestine alkaline phosphatase (CIP)
  • 1‐kb DNA ladder
  • Appropriate yeast reporter strains for transformation (e.g., YB1 for G i‐coupled receptors)
  • YAPD medium (see recipe)
  • –URA agar plates (see recipe)
  • Drug selection agar plates (see recipe)
  • 0.1 M lithium acetate in TE buffer (see recipe), sterile
  • Herring sperm DNA (e.g., Promega, cat. no. D181A)
  • 40% (w/v) PEG 4000 (e.g., Sigma, cat. no. 95904‐1kg‐F) made in 0.1 M lithium acetate (see recipe), sterile
  • Dimethyl sulfoxide (DMSO)
  • –URA medium (see recipe)
  • PCR thermal cycler
  • UV illuminator
  • 37°, 42°, and 55°C water baths
  • Temperature‐adjustable incubator shaker
  • 30° and 37°C humidified cell culture incubator
  • Spectrophotometer
  • 50‐ml centrifuge tubes, sterile
  • 1.5‐ml microcentrifuge tubes, sterile
  • Sterile 14‐ml conical tubes
  • Sterile 250‐ml flasks
  • 100 × 15–mm sterile cell culture dishes

Basic Protocol 3: Yeast Mutant Library Screening and Liquid Yeast Growth Assays

  Materials
  • Vector plus insert transformants grown on drug selection agar plates (see protocol 2)
  • –URA medium (see recipe)
  • –URA agar plates (see recipe)
  • Liquid growth assay medium (see recipe)
  • Test compound
  • 96‐well, flat‐bottomed plate with lid, low attachment (e.g., Costar, cat. no. 3937)
  • Parafilm
  • Plate shaker
  • 25° and 30°C humidified cell culture incubator
  • 100 × 15–mm and 150 × 15–mm cell culture dishes, sterile
  • Multi‐channel pipettors (8‐channel and 12‐channel) and sterile pipet tips
  • Centrifuge with 96‐well plate adaptor
  • Microplate reader
  • 15‐ and 50‐ml tubes, sterile

Basic Protocol 4: Determine the Mutation Site(s) and Confirm the Pharmacological Profile of Selected Candidates

  Materials
  • Yeast cultures (see protocol 3, step 21)
  • –URA medium (see recipe)
  • QIAprep spin miniprep kit (Qiagen, cat. no. 27104) containing:
    • Buffer P1
    • Buffer P2
    • Buffer N3
    • Spin columns
    • PB buffer
    • PE buffer
    • EB buffer
  • LB agar plates with appropriate antibiotic (see recipe)
  • One Shot TOP10 competent cells (Invitrogen, cat. no. C4040‐10)
  • SOC medium (see recipe)
  • LB liquid with appropriate antibiotic (see recipe)
  • Restriction enzymes and 10× buffers
  • 1% agarose gel
  • Sterile 14‐ml conical tubes
  • 25°, 30°, and 37°C humidified cell culture incubators with shakers
  • Sterile 1.5‐ml microcentrifuge tubes
  • Acid‐washed glass beads, 425‐ to 600‐µm (e.g., Sigma, cat. no. G8772)
  • 42°C water bath
  • Electrophoresis apparatus and power source
  • Additional reagents and equipment for yeast transformations (see protocol 1)
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Figures

Videos

Literature Cited

Literature Cited
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   Armbruster, B.N., Li, X., Pausch, M.H., Herlitze, S., and Roth, B.L. 2007. Evolving the lock to fit the key to create a family of G protein–coupled receptors potently activated by an inert ligand. Proc. Natl. Acad. Sci. U.S.A. 104:5163‐5168.
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