Manipulating Gene Expression in Projection‐Specific Neuronal Populations Using Combinatorial Viral Approaches

Bryan B. Gore1, Marta E. Soden1, Larry S. Zweifel1

1 University of Washington, Seattle, Washington
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 4.35
DOI:  10.1002/0471142301.ns0435s65
Online Posting Date:  October, 2013
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Abstract

The mammalian brain contains tremendous structural and genetic complexity that is vital for its function. The elucidation of gene expression profiles in the brain, coupled with the development of large‐scale connectivity maps and emerging viral vector–based approaches for target‐selective gene manipulation, now allows for detailed dissection of gene‐circuit interfaces. This protocol details how to perform combinatorial viral injections to manipulate gene expression in subsets of neurons interconnecting two brain regions. This method uses stereotaxic injection of a retrograde transducing CAV2‐Cre virus into one brain region, combined with injection of a locally transducing Cre‐dependent AAV virus into another brain region. This technique is widely applicable to the genetic dissection of neural circuitry, as it enables selective expression of candidate genes, dominant‐negatives, fluorescent reporters, or genetic tools within heterogeneous populations of neurons, based upon their projection targets. Curr. Protoc. Neurosci. 65:4.35.1‐4.35.20. © 2013 by John Wiley & Sons, Inc.

Keywords: AAV; CAV; viral vector; neural circuit; stereotaxic surgery

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Using Combinatorial Viral Strategies to Study Gene‐Circuit Interfaces
  • Support Protocol 1: Stereotaxic Surgery for Viral Delivery to Brain
  • Support Protocol 2: Adeno‐Associated Virus Production
  • Support Protocol 3: Production of CAV2‐Cre
  • Support Protocol 4: Brain Sectioning
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Using Combinatorial Viral Strategies to Study Gene‐Circuit Interfaces

  Materials
  • 4% (v/v) Fluorogold (Fluorochrome)/0.9% (w/v) saline, or other appropriate dye
  • Wild‐type mouse, fluorescent reporter mouse (e.g., GFP or tdTomato at the Rosa26 genome locus), or Cre‐driver mouse (e.g., Jackson Laboratories or GENSAT), age and gender matched for targeting and experimental mice
  • Cre‐conditional or nonconditional adeno‐associated virus encoding reporter or gene of interest (AAV; 1 × 109 particles/µl suggested; protocol 3)
  • 4% (w/v) paraformaldehyde (PFA), pH 7.4 (see recipe)
  • Canine associated virus‐2 encoding Cre recombinase (CAV2‐Cre; protocol 4)
  • Mouse brain atlas (e.g., Paxinos and Franklin, ; Dong, ; http://www.brain‐map.org)
  • Additional reagents and equipment for carrying out stereotaxic injection ( protocol 2), perfusion fixation (unit 1.1), AAV virus production ( protocol 3), CAV2‐Cre virus production ( protocol 4), and tissue sectioning ( protocol 5).

Support Protocol 1: Stereotaxic Surgery for Viral Delivery to Brain

  Materials
  • Anesthesia (e.g., isoflurane)
  • Wild‐type mouse, fluorescent reporter mouse, or Cre‐driver mouse (e.g., Jackson Laboratories or GENSAT)
  • Topical anesthetic (e.g., 5% lidocaine cream)
  • Sterile water
  • Betadine
  • 0.9% (w/v) saline, sterile
  • Virus, dye, or tracer of interest
  • Sutures or surgical adhesive
  • Stereotaxic equipment (e.g., Kopf)
    • Sterotaxic frame
    • 40× microscope attachment for stereotaxic frame
    • Stereotaxic alignment indicator
    • Stereotaxic drill
    • Syringe holder
  • Electric razor
  • Sterile cotton swabs
  • Surgical tools:
    • Scalpel
    • Skin clamps
    • Forceps
  • 5‐µl injection syringe (e.g., Hamilton)
  • Microsyringe pump (e.g., World Precision Instruments)

Support Protocol 2: Adeno‐Associated Virus Production

  Materials
  • ∼5 × 106 HEK293T/17 cells, low‐passage frozen stock (American Type Culture Collection)
  • HEK culture media (see recipe)
  • 0.25% (w/v) trypsin‐EDTA (Life Technologies)
  • 2M CaCl 2, sterile
  • AAV shuttle vector with desired construct
  • pDG1 packaging vector
  • HEPES solution (see recipe)
  • Phosphate mix (see recipe)
  • Serum‐free HEK medium (see recipe)
  • Dry ice/95% (v/v) ethanol bath
  • 40% (w/v) sucrose in phosphate‐buffered saline (PBS; appendix 2A), sterile
  • 1.37 g/ml CsCl solution (see recipe for CsCl solutions)
  • 6× DNA loading buffer ( appendix 2A)
  • 1% (w/v) agarose gel
  • 0.5 µg/ml ethidium bromide
  • 1× Hanks’ balanced salt solution (HBSS; appendix 2A)
  • Biosafety cabinet
  • Cell culture incubator
  • 10‐ and 15‐cm tissue culture dishes
  • Hemacytometer
  • 50‐ and 15‐ml conical centrifuge tubes
  • Pasteur pipets, sterile
  • 37°C water bath
  • Clinical centrifuge
  • 25 × 89–mm ultracentrifuge tubes (e.g., Beckman)
  • Ultracentrifuge (e.g., Beckman with SW‐27 and VTi‐65 rotors), or equivalent
  • 13 × 51–mm heat‐seal ultracentrifuge tubes and sealer (e.g., Beckman)
  • 1.5‐ml microcentrifuge tubes
  • 0.6‐ml thin‐walled microcentrifuge tubes (e.g., Thermo Scientific)
  • 2‐ to 3‐liter container
  • 5‐ml syringe with 20‐G needle
  • 10,000 molecular weight cutoff dialysis cassettes, 0.5‐ to 3‐ml capacity (e.g., Pierce)
  • 0.2‐µm pore syringe filter
  • Parafilm
  • Additional reagents and equipment for carrying out agarose gel electrophoresis ( appendix 1N)

Support Protocol 3: Production of CAV2‐Cre

  Materials
  • ∼5 × 106 DK‐ZEO cells, frozen stock (available from the authors upon request)
  • DK‐ZEO culture medium (see recipe)
  • 0.25% (w/v) trypsin‐EDTA (Life Technologies)
  • CAV2‐Cre viral vector stock (available from the authors upon request)
  • Dry ice/95% (v/v) ethanol bath
  • CsCl solutions (see recipe)
  • Phosphate‐buffered saline (PBS), with CaCl 2 and MgCl 2 (see recipe)
  • Viral lysis solution: 0.1% (w/v) SDS/10 mM Tris·Cl, pH 7.4 (see appendix 2A)
  • Biosafety cabinet cell culture incubator
  • 10‐ and 15‐cm tissue culture dishes
  • Cell scraper
  • 50‐ml conical centrifuge tubes
  • Clinical centrifuge
  • 25 × 89–mm and 14 × 89–mm ultracentrifuge tubes (e.g., Beckman)
  • Pasteur pipets
  • 20‐G needle and 5‐ml syringe
  • Ultracentrifuge (e.g., Beckman with SW‐27 and SW‐41 rotors), or equivalent
  • PD‐10 columns (e.g., GE Life Sciences)
  • 1.5‐ml microcentrifuge tubes
  • 37°C and 56°C water baths
  • Spectrophotometer

Support Protocol 4: Brain Sectioning

  Materials
  • Perfused mouse (e.g., see Basic Protocol)
  • 4% (w/v) paraformaldehyde (PFA), pH 7.4 (see recipe)
  • 30% (w/v) sucrose in PBS ( appendix 2A)
  • Freezing medium (e.g., O.C.T., TissueTek)
  • Dry ice/95% (v/v) ethanol bath
  • PBS ( appendix 2A)
  • Mounting medium (e.g., Fluoromount, Sigma‐Aldrich)
  • Small glass or plastic jar with a secure lid
  • Test tube rotator or rocker
  • Disposable embedding molds (Polysciences)
  • Rounded spatula or forceps
  • Cryostat
  • 24‐ or 48‐well plate
  • Paintbrush
  • Glass slides
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Figures

Videos

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Internet Resources
  http://jaxmice.jax.org
  A large number of transgenic mouse lines are available from Jackson Laboratories.
  http://www.gensat.org
  GENSAT maps the expression of genes in the central nervous system of the mouse.
  http://www.brain‐map.org
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  http://connectivity.brain‐map.org
  The AIBS Connectivity Atlas maps all the interconnections within the mouse brain.
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