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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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.
3. Set up the stereotaxic frame, mount the microsyringe injector onto the manipulator and connect the injector to the controller.
4. At....

Results

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. Transfection of dorsal horn neurons.

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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