In Ovo Electroporation in the Chicken Auditory Brainstem

Summary

Auditory brainstem neurons of avians and mammals are specialized for fast neural encoding, a fundamental process for normal hearing functions. These neurons arise from distinct precursors of embryonic hindbrain. We present techniques utilizing electroporation to express genes in the hindbrain of chicken embryos to study gene function during auditory development.

Abstract

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that the chicken embryo is an effective research model for studying basic biological functions of auditory system development. More recently, the chicken embryo has become particularly valuable in studying gene expression, regulation and function associated with hearing. In ovo electroporation can be used to target auditory brainstem regions responsible for highly specialized auditory functions. These regions include the chicken nucleus magnocellularis (NM) and nucleus laminaris (NL). NM and NL neurons arise from distinct precursors of rhombomeres 5 and 6 (R5/R6). Here, we present in ovo electroporation of plasmid-encoded genes to study gene-related properties in these regions. We show a method for spatial and temporal control of gene expression that promote either gain or loss of functional phenotypes. By targeting auditory neural progenitor regions associated with R5/R6, we show plasmid transfection in NM and NL. Temporal regulation of gene expression can be achieved by adopting a tet-on vector system. This is a drug inducible procedure that expresses the genes of interest in the presence of doxycycline (Dox). The in ovo electroporation technique – together with either biochemical, pharmacological, and or in vivo functional assays – provides an innovative approach to study auditory neuron development and associated pathophysiological phenomena.

Introduction

Fast neural encoding of sound is essential for normal auditory functions. These include sound localization abilities¹, speech in noise discrimination², and the comprehension of other behaviorally relevant communication signals³. Analogous neurons located in the auditory brainstem of both avians and mammals are highly specialized for fast neural encoding⁴. These include the chicken nucleus magnocellularis (NM), the nucleus laminaris (NL) and their mammalian analogs, the anteroventral cochlear nucleus (AVCN) and the medial superior olive (MSO), respectively^5....

Protocol

All procedures were approved by Northwestern University Institutional Animal Care and Use Committees, and carried out in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

1. Egg Handling

1. Purchase fertilized eggs from a local vendor. Store eggs at 13 °C in a refrigerator for no longer than 5 days prior to incubation. Embryo viability significantly decreases after 1 week.
2. Swab each egg with 70% ethanol before setting in the incubator.
3. Set 1-2 dozen(s) of eggs on their sides in an incubator with the temperature set to 38 °C at ~50% humidity. This e....

Results

We show here that in ovo electroporation permits gene expression in a normally developing biological system. Plasmid-encoded genes are focally injected into the neural tube overlying R5/R6. A schematic example of the electrode and pipette placements relative to important anatomical markers is shown in Figure 1A. The correct location of plasmid injection is confirmed 24 h after electroporation and shown in Figure 1B. The targeted injection of plas.......

Discussion

In ovo electroporation is a method of expressing or knocking down genes of interest in order to analyze in vivo gene function^8,9. In the chicken embryo, it is an innovative method for expressing plasmid-encoded genes into different auditory brainstem regions⁸. To ensure optimal expression, several critical steps are required. First, only inject embryos whose otocysts are clearly visible. If otocysts are not visible the em.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fertilized white leghorn chicken eggs	Sunnyside Inc. (Beaver Dam, WI)
Picospritzer	Parker Hannifin	052-0500-900	Picospritzer III, single or dual channel
Current/voltage stimulator	Grass Technologies	SD9	SD9
Microfil syringe needles	World Precision Instruments	MF28G67-5	28 Gauge, 67 mm Long, (Pack of 5)
Electrode holder	Warner Instruments	64-1280	MP Series: Non-Electrical Pressure Applications
Stimulating microelectrode	FHC	PBSA1075	PBSA1075
Air tank/regulator	NU Laboratory Services		Air dry 300 CF
Fast green	Sigma Aldrich	F7258-25G	F7258-25G
Clear plastic tape	Scotch	191
Doxycycline hyclate	Sigma Aldrich	D9891-1G
Egg refrigerator	Vissani Wine Refrigerator		13.3 - 16.1 °C (56-61° F)
Incubator	Hova-Bator		38 ° C (100 ° F), ~50% humidity
Dissection scope	Zeiss	4.35E+15	SteREO Discovery, V8 Microscope, 50.4X
Cold-light source	Zeiss	4.36E+15	CL6000 LED
Micromanipulators	Narishige Japan	Model: MM-3	2 Micromanipulators
Capillary tubes	Sutter Instrument	BF150-86-10	Thick-walled borosilicate (dimensions)
Syringes			1 mL, 3 mL
Needles	BD Precision Glide		27 G x 1 1/4, 19 G x 1 1/2
Forceps	Stoelting		No. 5 Super Fine Dumont
Egg holder	Custom Made		Clay base works as well
Micropipette puller	Sutter Instrument		Model P-97
Syringe filter	Ultra Cruz	sc-358811	PVDF 0.22 μm