Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

Summary

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

Abstract

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Introduction

Traditional methods for modifying neural gene expression require time-consuming and expensive germline manipulations. Alternative de novo approaches such as in utero electroporation or stereotaxic lentiviral injection yield faster results and are less costly but have the disadvantage of requiring complex surgical intervention^1-3. Furthermore, transgene expression has a limited spatial range with these methods. Herein, we describe a fast, easy, and economical method for widespread neuronal manipulation via intraventricular injection of adeno-associated virus (AAV) into the neonatal mouse brain. The method was first described by John Wolfe a....

Protocol

Perform all procedures and protocols involving animals in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The procedures described here were reviewed and approved by the Baylor College of Medicine Institutional Animal Care and Use Committee.

Replication-incompetent adeno-associated virus (AAV) vectors for transgene delivery in the rodent brain are approved for Biosafety Level 1 use. Refer to the CDC website for the US Government publication “Biosafety in Microbiology and Biomedical Laboratories (BMBL)” which details specific requirements regarding proper protection and virus ha....

Results

Successful intraventricular viral injection yields widespread and robust neuronal expression. Here we evaluated viral transduction using YFP or tdTomato fluorescent genes under the control of chicken beta actin promoter (CBA promoter). These constructs were packaged into AAV8 and injected into the lateral ventricles of neonatal (P0) ICR mice. High viral titers (10¹⁰ particles per hemisphere) resulted in dense labeling of the olfactory bulb, striatum, cerebral cortex, hippocampus and cerebellum (Figure .......

Discussion

We have described a versatile procedure for manipulating neuronal gene expression using AAV as a vehicle for widespread delivery into the neonatal mouse brain. Compared with other methods of neuronal transgenesis such as in utero electroporation¹ or stereotaxic intracranial injection^2,3, neonatal viral injection is relatively easy and simple. The basic procedure can be performed in minutes with only an ice bucket and a microliter syringe. Optimal survival and transgene expression can be attained by .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ICR outbred mice	Harlan	Hsd:ICR (CD-1)	This strain is also known as CD-1
FVB inbred mice	The Jackson Laboratory	1800	5-6 weeks of age
Nestlets	Lab Supply	NESTLETS
Shepherd shacks	Lab Supply	SS-mouse
High fat rodent chow	Purina Mills	PicoLab Mouse diet 20, #5058	This is our standard breeder chow
High fat rodent chow (alternative)	Harlan Laboratories	Teklad Global 19% protein rodent diet #2019S	If low phytoestrogen, autoclavable diet is needed
Injection syringe	Hamilton	7653-01	10 ml syringe
Injection needles	Hamilton	7803-04, RN 6PK PT4, 0.375"	32 G, for standard P0 injections
Metal plate for cryoanesthesia	McMaster Carr	8975K439	Raw aluminum plate, 6” x 12”, 0.25” thick, will need to be cut into 3 equal pieces and edges sanded by local machine shop
Small animal stereotaxic device with digital readout	David Kopf Instruments	Model 940
Universal syringe holder with needle support foot	David Kopf Instruments	Model 1772-F1
Neonatal frame	Stoelting	51625	Officially called a mouse and neonatal rat adaptor
Biohazard disposal bags with sterile indicator	VWR	14220-030	Important! – Check with local veterinary and environmental safety staff to learn your institute’s protocol for biohazard waste disposal