Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Summary

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

Abstract

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2´-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Introduction

Despite differences in morphology and cellular patterning between the mammalian and avian auditory sensory organ, the molecular factors and pathways responsible for sensory HC development are thought to be evolutionarily conserved ^1-3. The basilar papilla, which houses the auditory HCs and their surrounding SCs, develops as an outpocketing of the inner ear otocyst. Early on a pool of HC and SC progenitors is specified within the otic placode/otic cup neural-sensory competent domain (NSD). Fate-mapping data provide evidence that neuronal and sensory lineages are linked and arise from the NSD located in the antero-ventral region of the otic cup or otocyst in ....

Protocol

The eggs and unhatched embryos are cared for and treated ethically and humanely. All protocols for unhatched embryo use were approved by the Animal Care and Use Committee at the Johns Hopkins School of Medicine, Baltimore, Maryland.

1. Eggs and Preparation of Expression Constructs

1. Eggs:
   1. Incubate 3 - 4 dozen fertilized chicken eggs (Gallus gallus) lying flat on their sides at 37 °C.
      1. After 24 hr of incubation, pull 3 cc of albumen with a syringe from the round back end of the egg and seal the hole with clear tape. This creates an air chamber between the embryo and ....

Results

In this method paper plasmid DNA consisting of green fluorescent protein (GFP) expression cassettes was targeted into the developing chicken basilar papilla (BP) with optimized parameters of 12 V and 4 pulses with 100 msec pulse duration and intervals of 200 msec, yielding a ~50% embryo survival rate and efficiency of plasmid-DNA targeting into the BP. Fluorescent imaging of native GFP expression in developing embryos showed this method of electroporation preferentially targets the anteri.......

Discussion

The here described method of in ovo micro-electroporation is optimized for gene transfer into the developing auditory organ. It is compatible with plasmid DNA-based expression vectors typically used to manipulate gene function/expression. The timing of electroporation at E4 is optimal for investigations that focus on HC development in the auditory organ. The most critical steps are micro-injecting the DNA into the otic vesicle lumen without going too deep with the needle and damaging the otic vesicle (see

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sterile 1x PBS pH 7.4	gibco	10010-023
Fast Green FCF Powder	Sigma	F7252-5G
EdU Powder	Invitrogen	E10187
Click-IT EdU Alexa Fluor 555 Imaging Kit	Invitrogen	C10338
HiSpeed Plasmid Midi Kit (25)	Qiagen	12643
ECM 830 ElectroSquarePorator	BTX Harvard Apparatus
Banana to Micrograbber Cable Kit	BTX Harvard Apparatus	45-0216
Right Handed & Left Handed Micromanipulators	World Precision Instruments Inc.	M3301R & M3301L
Two 12 mm Magnetic Holding Device Stages with 7 inch vertical posts	World Precision Instruments Inc.	M10
Metal Steel Base Plate 12x24 inch	World Precision Instruments Inc.	5479
Scissors for Eggs 12 cm long curved, 12 mm extrafine blades; Spring Scissors	World Precision Instruments Inc.	14120
Micropippette Puller for pulling needles	Sutter Instrument Co.	P-97
2.5x2.5 mm Box Platinum Heating Filament	Sutter Instrument Co.
Glass Capillary Tubes/Needles/No Fiber/Borosil 1 mm	FHC	27-30-0
Hamilton Glass Syringe 100 μl	Hamilton	80601 Model 710LT
Mineral Oil (Heavy) for Hamilton Glass Syringe	Fisher Scientific	O122-1
Polyethylene Tubing for connecting the Glass Syringe and Glass Cappillary Needles/ Non Toxic	Becton Dickinson and Company (BD)	427420 Intramedic Clay Adams Brand
3 cc Disposable Syringes	Becton Dickinson and Company (BD)	309657
Disposable 21 Gauge Needles	Becton Dickinson and Company (BD)	305122
One pair of 2 mm Platinum Electrodes	Bulldog Bio. / Nepagene	CUY611P3-2
Electrode Holder	Bulldog Bio. / Nepagene	CUY580
One pair of Dumont fine forceps number 5	Fine Science Tools (FST)
Matte finish invisible tape for sealing eggs	Office Depot	520-928
Cotton-Tipped Swabs	Fisher Scientific	23-400-101
Sterile filter tips