Particle‐Mediated Gene Delivery In Vivo and In Vitro

Ning‐Sun Yang1, Joseph Burkholder1, Dennis McCabe1, Veronica Neumann1, Deb Fuller1

1 Auragen, Inc, Middleton, Wisconsin
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 12.6
DOI:  10.1002/0471142905.hg1206s12
Online Posting Date:  May, 2001
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Abstract

Particle‐mediated or “gene gun” technology has been developed as a nonviral method for gene transfer into various mammalian tissues. Gene delivery is achieved by physical force: a strong shock wave is generated that accelerates DNA‐coated gold particles to high speeds, providing them with the momentum needed to penetrate the targeted cells. This unit describes general procedures for in vivo and in vitro DNA and RNA transfections by particle‐mediated delivery. The and an alternate protocol address in vivo delivery to mouse skin. In vitro delivery to cryopreserved and adherent cells is also described.

     
 
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Table of Contents

  • Basic Protocol 1: In Vivo Particle‐Mediated Delivery of DNA to Mouse Skin by Gene Gun
  • Alternate Protocol 1: In Vivo Particle‐Mediated Delivery of RNA to Mouse Skin by Gene Gun
  • Alternate Protocol 2: In Vitro Particle Delivery to Cryopreserved or Nonadherent Cells
  • Alternate Protocol 3: In Vitro Particle Delivery to Adherent Cell Cultures
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: In Vivo Particle‐Mediated Delivery of DNA to Mouse Skin by Gene Gun

  Materials
  • 0.05 M spermidine (melt spermidine under warm water and dilute to 0.05 M in water; store at room temperature ≤1 month)
  • ∼1 mg/ml purified plasmid DNA in TE buffer ( appendix 2D) or water
  • 1 M calcium chloride
  • 100% ethanol
  • Compressed helium gas, grade 4.7 (e.g., Airco or AGA gas)
  • 10% bleach
  • Mice
  • Gene gun system:
  •  ∼1‐ to 3‐µm gold particles
  •  Tefzel tubing
  •  Tubing prep unit
  •  Gene gun
  • Tube cutter
  • Ultrasonic cleaner: e.g., Fisher FS3, 70 W, 55 kHz
  • Personal protective gear, including hearing and eye protection
  • Oster A5 clippers with no. 40 blade (Fisher)
  • Additional reagents for animal anesthesia (units 13.2 & 13.4)
  • CAUTION: Observe all regulations regarding use of research animals and recombinant DNA materials, and follow approved procedures concerning animal use as outlined in your institution's animal care and use policy.

Alternate Protocol 1: In Vivo Particle‐Mediated Delivery of RNA to Mouse Skin by Gene Gun

  • 7 µg RNA in ≤25 µl volume
  • 5 M ammonium acetate
  • 100% isopropanol

Alternate Protocol 2: In Vitro Particle Delivery to Cryopreserved or Nonadherent Cells

  • Primary tumor cell line of interest (cryopreserved; appendix 3G) or nonadherent cells (such as PBMC)
  • Appropriate culture medium, 37°C
  • 1 M HEPES in normal saline (Bio‐Whittaker)
  • Wide‐bore pipet tips (e.g., Bio‐Rad)
  • Additional reagents for mammalian cell culture and counting of viable cells using a hemacytometer ( appendix 3G)
NOTE: All reagents and equipment coming into contact with live cells must be sterile, and proper sterile technique should be followed accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Alternate Protocol 3: In Vitro Particle Delivery to Adherent Cell Cultures

  • Adherent cell line of interest, in culture or cryopreserved ( appendix 3G)
  • Appropriate culture medium
  • Additional reagents and equipment for mammalian cell culture, trypsinization, and counting of viable cells using a hemacytometer ( appendix 3G)
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Figures

Videos

Literature Cited

Literature Cited
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