Reducing Gene Expression in the Brain via Antisense Methods

Abdel‐Mouttalib Ouagazzal1, James M. Tepper2, Ian Creese2

1 United Medical and Dental Schools, Guy's Hospital, London, 2 Rutgers University Center for Molecular and Behavioral Neuroscience, Newark, New Jersey
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 5.4
DOI:  10.1002/0471142301.ns0504s00
Online Posting Date:  May, 2001
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Abstract

This unit presents protocols that employ antisense oligodeoxynucleotides to reduce expression of target proteins in the brain. These oligonucleotides are generally designed to inhibit synthesis of a specific protein by hybridization to its mRNA. Because oligonucleotides show very poor penetration into the central nervous system (CNS) after systemic administration, they are either injected into the cerebrospinal fluid (CSF) or infused directly into the brain parenchyma. In this unit, the procedure most commonly used for delivering oligonucleotides continuously into CSF is outlined. In addition, a procedure is described for continuous infusion of oligonucleotides into a specific brain region, using the substantia nigra as an example.

     
 
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Table of Contents

  • Basic Protocol 1: Chronic Intracerebroventricular Infusion of Antisense Oligodeoxynucleotides
  • Basic Protocol 2: Chronic Intranigral Infusion of Antisense Oligodeoxynucleotides
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Chronic Intracerebroventricular Infusion of Antisense Oligodeoxynucleotides

  Materials
  • Oligodeoxynucleotides (custom‐synthesized, 19‐mer phosphorothioate‐modified, and HPLC‐purified; e.g., Oligos Etc.)
  •  Dopamine D 2 antisense oligonucleotide complementary to nucleotides 4 through 22 of the rat D 2 mRNA (5′‐AGGACACGGTTCAGTGTC‐3′)
  •  Control oligonucleotide (scrambled sequence of the bases in the D 2 antisense oligonucleotide; 5′‐AGAACGGCATTAGTGGGT‐3′)
  • 0.9% NaCl, pH 6.5 to 7 (sterile)
  • Male Sprague‐Dawley rats (Zivic‐Miller) averaging 260 g at time of surgery
  • Anesthetic solution (see recipe)
  • Dental cement and acrylic solvent (Plastics One)
  • Disinfectant (e.g., Betadine)
  • Alzet brain infusion kit (Alza) consisting of:
  •  L‐shaped cannulas
  •  Catheter tubes
  •  Depth‐adjustment spacers
  • Alzet microosmotic pumps and flow moderators (model 1003D; Alza)
  • Syringes with 27‐G needles
  • Stereotaxic surgery materials:
  •  Small animal clippers
  •  Instruments for small animal surgery (e.g., scalpel, scissors, spatula, hemostat)
  •  Hot glass bead dry sterilizer (Stoelting)
  •  Stereotaxic apparatus (David Kopf Instruments)
  •  Dual‐pipe (fiber‐optic) illuminators (Stoelting)
  •  Sterile cotton swabs
  •  Rat brain atlas (Paxinos and Watson, )
  •  Stainless steel bit (part number D‐HSD‐56), pin vise (part number D‐PV‐5), and stainless steel screws (0‐80 machine screws; Small Parts)
  •  Autoclip wound clips and Autoclip applier (Stoelting)
  • Heating pad or infrared lamp
  • Additional reagents and equipment for measuring DNA concentration (CPMB APPENDIX and appendix 1A in this manual)
NOTE: Perform all steps at room temperature.NOTE: Use sterile technique during preparation of the oligodeoxynucleotide solutions as well as during the handling and filling of microosmotic pumps. All surgical instruments must be sterile and procedures are to be performed in a sterile manner unless otherwise specified.

Basic Protocol 2: Chronic Intranigral Infusion of Antisense Oligodeoxynucleotides

  Materials
  • Male Sprague‐Dawley rats (Zivic‐Miller) averaging 260 g at time of surgery
  • Anesthetic solution (see recipe)
  • Dental cement and acrylic solvent (Plastics One)
  • Disinfectant (e.g., Betadine)
  • Oligodeoxynucleotides (custom synthesized, 19‐mer phosphorothioate‐modified, and HPLC‐purified; e.g., Oligos Etc.)
  •  Dopamine D 2 antisense oligonucleotide complementary to nucleotides 4 through 22 of the rat D 2 mRNA (5′‐AGGACACGGTTCAGTGTC‐3′)
  •  Control oligonucleotide (scrambled sequence of the bases in the D 2 antisense oligonucleotide; 5′‐AGAACGGCATTAGTGGGT‐3′)
  • 0.9% NaCl, pH 6.5 to 7 (sterile)
  • 100% ethanol
  • Sterile distilled H 2O
  • Syringes with 27‐G needles
  • Stereotaxic surgery materials:
  •  Small animal clippers
  •  Instruments for small animal surgery (e.g., scalpel, scissors, spatula)
  •  Hot glass bead dry sterilizer (Stoelting)
  •  Stereotaxic apparatus (David Kopf Instruments)
  •  Dual‐pipe (fiber‐optic) illuminators (Stoelting)
  •  Sterile cotton swabs
  •  Rat brain atlas (Paxinos and Watson, )
  •  Stainless steel bit (part number D‐HSD‐56), pin vise (part number D‐PV‐5), and  stainless steel screws (0‐80 machine screws; Small Parts)
  •  Autoclip wound clips and Autoclip applier (Stoelting)
  • Cannulas for infusion system:
  •  26‐G guide cannula cut 7 mm below pedestal (Plastics One)
  •  32‐G dummy cannula (stylet) that fits exactly into guide cannula (Plastics One)
  •  33‐G injector cannula 1‐mm longer than guide cannula (Plastics One)
  • Chronic animal infusion system:
  •  Single connector with spring (∼40 cm; Plastics One)
  •  Single‐channel swivel (Instech Laboratories)
  •  Standard PE50 catheter tubing (Becton Dickinson Primary Care Diagnostics)
  •  25‐µl Hamilton syringe
  •  Microliter syringe pump (Instech Laboratories or Harvard Apparatus)
  • 5‐ml syringes
  • Circular Plexiglas cages (diameter 28 cm; height 30 cm)
  • Additional reagents and equipment for measuring DNA concentration (CPMB APPENDIX and appendix 1A in this manual)
NOTE: Perform all steps at room temperature.NOTE: Use sterile technique during preparation of the oligonucleotide solutions as well as during the handling of the chronic infusion system components. All surgical instruments must be sterile and procedures are to be performed in a sterile manner unless otherwise specified.
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Literature Cited

Literature Cited
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Key Reference
   Hagg, T. 1994. Continuous central nervous system infusion with Alzet osmotic pumps. In Methods in Neurosciences,Vol. 21: Providing Pharmacological Access to the Brain: Alternate Approaches (T.R. Flanagan, D.F. Emerich, and S.R. Winn, eds.) pp. 201‐213. Academic Press, San Diego.
  Describes the use of Alzet osmotic pumps for continuous infusion into CSF or directly into the parenchyma.
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