Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Summary

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

Abstract

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.

The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

Protocol

1. Introduction

Creation of transgenic animals is a powerful method of investigation of gene functions in living animals¹ and for unraveling disease mechanism^2,3 as well as for manipulating properties of cells⁴. However, the procedure is rather laborious, extremely time consuming and expensive, thus warranting the use of alternative gene delivery methods such as viral injection⁵, neonatal ventricular injection with electroporation⁶ and in utero electroporation^7,8. The method of postnatal non-ventricular injection and electroporation has a unique set of advantages: use....

Discussion

Methods of gene delivery into living rodent brain are well established for in utero electroporation^7,8,11,12 and, more recently, for postnatal electroporation⁶. However, these methods are based on intraventricular injection of the plasmid DNA, which may be limiting for several applications. For example, these methods do not allow targeting cells in certain brain areas such as hippocampus, nor transfection of such non-migratory cell types as cortical astrocytes. Non-ventricular injection cou.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2A-sa dumb Tweezers, 115mm	equipment	XYtronic	XY-2A-SA	Treat with 70% ethanol for disinfection before use in surgical manipulations
Biological Temperature Controller with stainless steel heating pad	equipment	Supertech	TMP-5b
Borosilicate tube with filament	material	Sutter Instrument Co.	BF120-69-10	Glass needle
Disposable drills	material	Meisinger	HP 310 104 001 001 008
Dulbeco’s PBS 10X	reagent	Sigma-Aldrich	D1408
Dumont #5 forceps, 110 mm	equipment	Fine Science Tools	91150-20	Treat with 70% ethanol for disinfection before use in surgical manipulations
Ealing micr–lectrode puller	equipment	Ealing	50-2013	Vertical electrode glass puller
Ethilon monofil polyamide 6-0 FS-3 16 mm 3/8c	material	Johnson & Johnson	EH7177H	Surgical threads
Exmire micro syringe 10.0 ml	equipment	Exmire	MS*GLLX00	Gas-tight syringe
Fast Green	reagent	Sigma-Aldrich	F7252
Forceps electrodes	equipment	BEX	LF650P3	Treat with 70% ethanol for disinfection prior to use
Foredom drill control	equipment	Foredom	FM3545	Surgical drill power supply and control. Currently available analogue is micromotor kit K.1070 (Foredom)
Foredom micro motor handpiece	equipment	Foredom	MH-145	Currently available analogue is micromotor kit K.1070 (Foredom)
Gas anesthesia platform for mice	equipment	Stoelting Co.	50264	Assembled on stereotaxic instrument
Isoflurane	reagent	Baxter Internationl Inc.	FDG9623
Micro dressing forceps, 105 mm	equipment	Aesculap	BD302R	Treat with 70% ethanol for disinfection before use in surgical manipulations
Microfil	material	World Precision Instruments, Inc.	MF34G-5	Micro syringe filling capillaries
Mineral oil	reagent	Sigma-Aldrich	M8410
NanoFil Syringe 10 microliter	equipment	World Precision Instruments, Inc.	NANOFIL	Hamilton syringe
plasmid CAG-EGFP	reagent			Extracted and purified with EndoFree Plasmid Maxi Kit (Qiagen) and dissolved in nuclease free water to concentration 1.5 mg/ml
Pulse generator CUY21Vivo-SQ	equipment	BEX	CUY21Vivo-SQ
Schiller electrode gel	reagent	Schiller AG	2.158000	Conductive gel
Small animal stereotaxic instrument	equipment	David Kopf Instruments	900
St–lting mouse and neonatal rat adaptor	equipment	Stoelting Co.	51625	Assembled on stereotaxic instrument.Treat earbars with 70% ethanol for disinfection before use in surgical manipulations
Student iris scissors, straight 11.5 cm	equipment	Fine Science Tools	91460-11	Treat with 70% ethanol for disinfection before use in surgical manipulations
Sugi absorbent swabs 17 x 8 mm	material	Kettenbach	31602	Surgical tampons
UMP3 microsyringe pump and Micro 4 microsyringe pump controller	equipment	World Precision Instruments, Inc.	UMP3-1	Microinjector and controller
Univentor 400 Anesthesia Unit	equipment	Univentor	8323001