Gene Transfer to the CNS Using Recombinant Adeno‐Associated Virus

Lorelei Stoica1, Seemin S. Ahmed2, Guangping Gao3, Miguel Sena‐Esteves3

1 Department of Neurology, University of Massachusetts Medical School, Worcester, Masssachusetts, 2 Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Masssachusetts, 3 Co‐corresponding authors
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 14D.5
DOI:  10.1002/9780471729259.mc14d05s29
Online Posting Date:  May, 2013
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Abstract

Recombinant adeno‐associated virus (rAAV) vectors are great tools for gene transfer due to their ability to mediate long‐term gene expression. rAAVs have been used successfully as gene transfer vehicles in multiple animal models of CNS disorders, and several clinical trials are currently underway. rAAV vectors have been used at various stages of development with no apparent toxicity. There are multiple ways of delivering AAV vectors to the mouse CNS, depending on the stage of development. In neonates, intravascular injections into the facial vein are often used. In adults, direct injections into target regions of the brain are achieved with great spatiotemporal control through stereotaxic surgeries. Recently, discoveries of new AAV vectors with the ability to cross the blood brain barrier have made it possible to target the adult CNS by intravascular injections. Curr. Protoc. Microbiol. 29:14D.5.1‐14D.5.18. © 2013 by John Wiley & Sons, Inc.

Keywords: AAV; intracranial; intravascular; neonate stereotaxic

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Craniotomy and Stereotaxic Injection of AAV Vectors in the Brain of Adult Mice
  • Basic Protocol 2: Tail Vein Injection of AAV Vectors in Adult Mice
  • Basic Protocol 3: Intravenous Delivery of rAAV Vectors to Neonatal Mice
  • Support Protocol 1: Collection and Embedding of Mouse Brain for Histological Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Craniotomy and Stereotaxic Injection of AAV Vectors in the Brain of Adult Mice

  Materials
  • 0.5 M NaOH, sterile
  • Sterile water
  • Recombinant adeno‐associated virus (rAAV) vector stock
  • 1× phosphate‐buffered saline (PBS), filter sterilized
  • Mouse to be injected
  • Ketamine/xylazine anesthesia solution (see recipe)
  • Ophthalmic ointment (e.g., Puralube, Webster, cat. no. 07‐888‐2572)
  • Povidone‐iodine prep pads
  • Alcohol prep pads, 70% isopropyl alcohol
  • Ketoprofen analgesia solution (see recipe)
  • Stereotaxic frame (Stoelting, cat. no. 51730)
  • Injector (Ultra Micro Pump III, World Precision Instruments)
  • Syringe pump controller (World Precision Instruments, cat. no. SYS‐MICRO4)
  • 10‐µl gastight Hamilton syringe (Hamilton, cat. no. 7653‐01), autoclaved
  • 33‐G steel needle (Hamilton, cat. no. 7762‐06), autoclaved
  • Autoclave pouch for surgical tools
  • 1‐ml insulin syringes with permanent 28‐G needles (Kendal Monoject, cat. no. 1188128012)
  • Small mouse shaver
  • Sterile disposable scalpels (Dynarex, cat. no. 4111)
  • Sterile cotton swabs (Puritan, cat. no. 25‐806 1WC)
  • Stereotaxic Atlas (Paxinos and Franklin, )
  • Hand‐held drill with autoclaved drill bits
  • Sterile wound reflex clip stapler (Kent Scientific, cat. no. INS500345) with sterile 9‐mm wound reflex clips (World Precision Instruments, cat. no. 500346) and clip remover (Kent Scientific, cat. no. INS500347)
  • Circulating‐water warming pad

Basic Protocol 2: Tail Vein Injection of AAV Vectors in Adult Mice

  Materials
  • Recombinant adeno‐associated virus (rAAV) vector stock
  • 1× phosphate‐buffered saline (PBS), filter sterilized
  • Mouse to be injected
  • Alcohol prep pad, 70% isopropyl alcohol
  • 1‐ml allergy syringe with 27‐G, 3/8‐in. needle (Becton Dickinson, cat. no. 305541)
  • Infrared heat lamp
  • Mouse restrainer for tail vein injection (e.g., Kent Scientific, cat. no. HLD‐MS‐T)
  • Sterile gauze

Basic Protocol 3: Intravenous Delivery of rAAV Vectors to Neonatal Mice

  Materials
  • Recombinant adeno‐associated virus (rAAV) vector stock
  • 1× phosphate‐buffered saline (PBS), filter sterilized
  • Neonatal mouse to be injected
  • Isofluorane
  • 70% (v/v) isopropyl alcohol
  • 3/10‐cc syringe with 31‐G, 8‐mm‐long needle (e.g., Becton Dickinson, Ultra‐Fine II short needle insulin syringe)
  • Anesthesia chamber for neonate: made from 50‐ml Falcon tube stuffed with tissue paper
  • Sterile gauze

Support Protocol 1: Collection and Embedding of Mouse Brain for Histological Analysis

  Materials
  • 4% (w/v) paraformaldehyde (PFA) solution (see recipe)
  • 1× phosphate‐buffered saline (PBS)
  • Mouse to be perfused
  • Ketamine/xylazine anesthesia solution (see recipe)
  • 30% (w/v) sucrose in PBS
  • 2‐O‐Methylbutane
  • Dry ice
  • Optimal cutting temperature (OCT) medium (e.g., Thermo Scientific NEG50)
  • Peristalic pump (Fisher, cat. no. 13‐876)
  • Three‐way stopcock
  • Silicone tubing
  • Cannulated needle: scalp vein (butterfly) set with 1/2‐in., 25‐G needle (Exel, cat. no. 26708)
  • Surgical scissors
  • Retractor
  • Flat tweezers
  • Curved hemostatic forceps
  • 15‐ml plastic conical tube
  • Plastic disposable embedding mold (Electron Microscopy Sciences, cat. no. 70182)
NOTE: Perform all steps in a well‐ventilated chemical fume hood.
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Figures

Videos

Literature Cited

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