DNA Immunization

Shixia Wang1, Shan Lu1

1 University of Massachusetts Medical School, Worcester, Massachusetts
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 18.3
DOI:  10.1002/9780471729259.mc1803s31
Online Posting Date:  November, 2013
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DNA immunization was discovered in early 1990s, and its use has been expanded from vaccine studies to a broader range of biomedical research areas, such as the generation of high‐quality polyclonal and monoclonal antibodies as research reagents. In this unit, three common DNA immunization methods are described: needle injection, electroporation, and gene gun. In addition, several common considerations related to DNA immunization are discussed. Curr. Protoc. Microbiol. 31:18.3.1‐18.3.24. ©2013 by John Wiley & Sons, Inc.

Keywords: DNA vaccine; immunization; electroporation; gene gun

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: DNA Immunization by Needle Injection
  • DNA Immunization via Electroporation
  • Basic Protocol 2: Electroporation Using Caliper Electrode
  • Alternate Protocol 1: Electroporation Using Needle Electrode Electroporator
  • Basic Protocol 3: DNA Immunization Using a Gene Gun to Deliver Gold Beads Coated with DNA Plasmids
  • Support Protocol 1: Preparation of Gold Beads Coated with DNA Vaccine Plasmids
  • Support Protocol 2: Preparation of Gene Gun Tubing and Shots
  • Commentary
  • Literature Cited
  • Tables
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Basic Protocol 1: DNA Immunization by Needle Injection

  • Desired animal species (mice or rabbits)
  • Purified DNA vaccine plasmids in 0.9% (w/v) NaCl (pH 7.0 to 7.2), with a proper DNA concentration, usually in the range of 1 to 3 mg/ml
  • Mouse restrainers (optional; appendix 3N)
  • 0.5‐ or 1‐ml disposable syringes with 26‐ or 30‐G needles
  • Rabbit restrainers (optional; Donovan and Brown, )
  • Butterfly infusion set (BD Vacutainer Safety‐Lok Blood Collection Set, 23‐G × 3/4 in. × 12 in.) for rabbit blood collection (also see Donovan and Brown, )
  • 20‐ml syringes for rabbit blood collection (also see Donovan and Brown, )
  • Vacutainer with separation gel for serum collection (BD‐Vacutainer SST, 10 ml) for rabbit blood collection (also see Donovan and Brown, )
  • Additional reagents and equipment for anesthesia of mice and rabbits (see Strategic Planning), animal restraint (Donovan and Brown, ), injection of mice ( appendix 3N), injection of rabbits (Donovan and Brown, ), and blood collection from mouse or rabbit ( appendix 3N or Donovan and Brown, , respectively)

Basic Protocol 2: Electroporation Using Caliper Electrode

  • Desired animal species: mice or rabbits
  • Purified, sterile DNA vaccine plasmid in 0.9% (w/v) NaCl (pH 7.0 to 7.2) with a proper DNA concentration, usually 1 to 3 mg/ml
  • Animal clippers
  • Laboratory vacuum cleaner
  • Electroporator device (such as SCIENTZ‐2C from Scientz, http://www.scientzbio. com/)
  • Additional reagents and equipment for anesthesia (see Strategic Planning), needle injection of mice or rabbits ( protocol 1; also see appendix 3N and Donovan and Brown, ), and blood collection from mouse or rabbit ( appendix 3N or Donovan and Brown, , respectively)

Alternate Protocol 1: Electroporation Using Needle Electrode Electroporator

  Additional Materials (also see protocol 2)
  • Electroporator device (two are available commercially for pre‐clinical studies):
    • The BTX AgilePulse is designed for vaccine development and gene therapy applications and provides i.d. or i.m. electroporation options; the AgilePulse In Vivo System can be purchased with software supporting either i.d. or i.m. applications (see YouTube training video for BTX Agile Pulse at http://www.youtube.com/watch?v=89LCc_eki10)
    • The Teresa Gene Delivery Device made by Shanghai Teresa Bio‐Tech Co. (http://www.teresabio.com) has two models with focus on i.m. delivery of DNA vaccines.

Basic Protocol 3: DNA Immunization Using a Gene Gun to Deliver Gold Beads Coated with DNA Plasmids

  • Desired animal species: mice or rabbits
  • Compressed helium gas tank with helium regulator
  • Animal clippers
  • Laboratory vacuum cleaner
  • Helios gene gun (BioRad)
  • Cartridges prepared in Support Protocols protocol 51 and protocol 62
  • Barrel liner
  • Cartridge holder
  • Helium hose
  • O‐ring
  • Additional reagents and equipment for anesthesia (see Strategic Planning)

Support Protocol 1: Preparation of Gold Beads Coated with DNA Vaccine Plasmids

  • Gold beads of 0.5 to 5 µm diameter (BioRad, cat. no. 165‐2263); for most animal species, 1‐µm gold beads should be used as the first step
  • 100 mM nuclease‐free spermidine (Sigma, cat. no. S‐0266)
  • Plasmid DNA expressing the gene of interest in TE) buffer, pH 8.0 ( appendix 2A), at a concentration of 1 to 5 mg/ml
  • 2 M CaCl 2 solution, sterile (nuclease‐free)
  • Dehydrated ethanol, 200 proof (e.g., Spectrum, cat. no. ET107; https://www.spectrumchemical.com/)
  • 2.0‐ml microcentrifuge tubes
  • Water bath–type sonicator (Aquasonic Model 50T, VWR Scientific)
  • 22‐ml glass scintillation vials and Teflon caps

Support Protocol 2: Preparation of Gene Gun Tubing and Shots

  • Compressed nitrogen gas tank with nitrogen regulator
  • Gold beads prep from protocol 5
  • Tefzel tubing, outer diameter 0.127 in., inner diameter 0.093 in. (BioRad, cat. no. 165‐2424; includes syringe kit, Tefzel tubing, tubing cutter, optimization kit)
  • Caps for tubing (supplied with Tefzel tubing kit from BioRad)
  • Tubing prep station (BioRad)
  • 10‐ml syringes with Tefzel tubing adaptor (5 mm o.d. × 3 mm i.d. soft rubber tubing)
  • Water bath–type sonicator
  • Tubing cutter (BioRad)
  • 22‐ml glass scintillation vials and Teflon caps
  • Desiccant capsules
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