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Abstract

Introduction

Protocol

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Neuroscience

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Published: March 18th, 2013

DOI:

10.3791/50313

1Département Nociception et Douleur, Institut des Neurosciences Cellulaires et Intégratives, Centre National de la Recherche Scientifique (CNRS), 2Departments of Anesthesiology and Pharmacology, Columbia University , 3Department of Anesthesiology, Niigata University Graduate School of Medical and Dental Sciences

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Advanced technologies of conditional gene manipulation in the mouse enable multifaceted approaches to the exploration of synaptic pathways and functional connections in the central nervous system. Transgenes may be regulated by small-molecule effectors such as doxycycline acting on a tetracycline-controlled transactivator, which can be designed to function as a repressor or an activator of gene transcription, or tamoxifen recognizing a mutated ligand-binding domain of the estrogen receptor 1. Irreversible transgene modification is commonly achieved by deoxyribonucleic acid (DNA) recombinases. Cre (causes recombination) and Flp (flippase recombination enzyme....

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The surgical procedure described has been approved by the Institutional Animal Care and Use Committee (IACUC) of Columbia University.

1. Preparation of Equipment and Virus Particle Suspension

  1. Clean and disinfect the equipment, sterilize the surgical instruments and the V notch spikes that will be used to fix vertebra L1.
  2. Pull and bevel glass pipettes. We use pipettes that have a tip diameter of 40 μm and are beveled at an angle of 20°. Sterilize the glass pipettes........

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Successful transfection yields robust gene expression in neurons of the injected dorsal horn (Figure 1), sparing the dorsal horn of the contralateral side, the ventral horn and the dorsal root ganglia.

Figure 1
Figure 1. Transfection of dorsal horn neurons. (A) Expression of the fluorescent reporter eGfp (green) in the left dorsal horn of the L4 spinal cord, two weeks after the s.......

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Stereotaxic vector injection allows targeting spinal cord neurons for applications such as neuronal network mapping based on transsynaptic virus spreading 6,7 or optogenetic dissection 8, axon guidance during regeneration from injury 9,10, or gene therapy for the prevention or treatment of neurodegeneration 11,12. Viral vectors have been used for gene manipulation in the spinal cord to study somatosensory, motor and autonomic pathways 9,10,13-15. The mouse is the mod.......

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We thank Bakhos A. Tannous, Ph.D., Director of Vector Development and Production in the Neuroscience Center of Massachusetts General Hospital, Charlestown, Massachusetts, for providing us with the rAAV-eGfp vector, and John Whang for technical assistance. This work was supported by grant R01 NS050408 (to J.S.) from the National Institute of Neurological Disorders and Stroke.

....

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Name Company Catalog Number Comments
Material Name Company Catalogue Number
Spinal base plate David Kopf Instruments 912
Small animal stereotaxic instrument David Kopf Instruments 900
Mouse gas anesthesia head holder David Kopf Instruments 923-B
Adjustable base mounts David Kopf Instruments 982
V notch spikes David Kopf Instruments 987
Small animal temperature control system David Kopf Instruments TCAT-2LV
Adson forceps Fine Science Tools 11006-12
Laminectomy forceps Fine Science Tools 11223-20
UltraMicroPump (one) with SYS-Micro4 Controller World Precision Instruments UMP3-1
Microsyringe, 65RN Hamilton 7633-01
RN compression fitting, 1 mm Hamilton 55750-01
Borosilicate glass capillaries World Precision Instruments 1B100F-4
Microgrinder Narishige EG-44

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