Cre Activated and Inactivated Recombinant Adeno‐Associated Viral Vectors for Neuronal Anatomical Tracing or Activity Manipulation

Arpiar Saunders1, Bernardo L. Sabatini1

1 Department of Neurobiology, Harvard Medical School, Howard Hughes Medical Institute, Boston, Massachusetts
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 1.24
DOI:  10.1002/0471142301.ns0124s72
Online Posting Date:  July, 2015
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Recombinant adeno‐associated viruses (rAAVs) transcriptionally activated by Cre recombinase (Cre‐On) are powerful tools for determining the anatomy and function of genetically defined neuronal types in transgenic Cre driver mice. Here we describe how rAAVs transcriptionally inactivated by Cre (Cre‐Off) can be used in conjunction with Cre‐On rAAVs or genomic Cre‐reporter alleles to study brain circuits. Intracranial injection of Cre‐On/Cre‐Off rAAVs into spatially intermingled Cre+ and Cre neurons allows these populations to be differentially labeled or manipulated within individual animals. This comparison helps define the unique properties of Cre+ neurons, highlighting the specialized role they play in their constituent brain circuits. This protocol touches on the conceptual and experimental background of Cre‐Off rAAV systems, including caveats and methods of validation. © 2015 by John Wiley & Sons, Inc.

Keywords: AAV; rAAV; Cre‐lox; Cre‐dependent virus; viral tracer

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Preparation of Cre‐ON/OFF rAAVs for Intracranial Injection
  • Basic Protocol 2: Fixed Tissue Validation Techniques for Cre‐OFF rAAV Expression
  • Basic Protocol 3: Immunohistochemistry on Free‐Floating Brain Slices
  • Reagents and Solutions
  • Commentary
  • Figures
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Basic Protocol 1: Preparation of Cre‐ON/OFF rAAVs for Intracranial Injection

  • Optional: 1× PBS ( appendix 2A) with 350 mM NaCl and 5% (w/v) sorbitol (for dilution)
  • Microcentrifuge
  • PCR tubes (individual or strip)
  • Stereotactic injection setup and pipet (units 1.20 & 4.35)

Basic Protocol 2: Fixed Tissue Validation Techniques for Cre‐OFF rAAV Expression

  • 4% paraformaldehyde (PFA; ∼10 ml/mouse; see recipe)
  • 1× PBS ( appendix 2A)
  • Mounting media (for mounting and imaging tissue sections; e.g., Life Technologies, cat. no. P36935)
  • Fixative‐only dissection tools (for brain removal)
  • 20‐ml glass scintillation vials (for brain storage)
  • 24‐well plate
  • Supplies for mounting and imaging tissue sections
    • Small paint brush
    • Glass slides
    • Cover slips
    • Fluorescent microscope
  • Additional reagents and equipment for intracardiac perfusion fixation and fixed tissue sectioning (unit 1.1) and for stereotactic injections (units 1.20 & 4.35)
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.NOTE: All reagents and equipment coming into contact with live cells must be sterile, and proper sterile technique should be followed accordingly.

Basic Protocol 3: Immunohistochemistry on Free‐Floating Brain Slices

  • 1× PBS ( appendix 2A)
  • Blocking/dilution buffer (see recipe)
  • Primary antibody (e.g., mouse monoclonal Cre recombinase; Millipore, cat. no. MAB 3120)
  • Secondary antibody (e.g., donkey anti‐mouse Alexa Fluor 647; Life Technologies, cat. no. A‐31571)
  • Mounting media (for mounting and imaging tissue sections; e.g., Life Technologies, cat. no. P36935)
  • Optional: Nuclear counterstain (for mounting and imaging tissue sections)
  • 24‐well plate
  • Paint brush
  • Benchtop shaker
  • Aluminum foil
  • Supplies for mounting and imaging tissue sections
    • Glass slides
    • Cover slips
    • Fluorescent microscope
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Internet Resources
  DO, FAS, and Cre‐Switch rAAVs available from Addgene.
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