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), respectively⁵. However, developmental mechanisms regulating fast neural encoding are poorly understood in the auditory brainstem. Therefore, it is advantageous to study specific genes that are responsible for fast neural encoding in order to better understand their expression, regulation and function in auditory development.

The developing chicken embryo is an effective and well-established research tool to study basic biological questions of auditory system development^6,7. Recent molecular advances have addressed these biological questions in the developing chicken embryo by expressing or knocking down genes of interest in order to analyze in vivo gene function^8,9. Investigating the regulatory role of specific genes is a significant advancement in understanding pathologies associated with auditory deficits. Here, we present in ovo electroporation of plasmid-encoded genes into the chicken auditory brainstem where fast neural encoding of sound occurs¹⁰. By targeting auditory neural progenitor regions associated with rhombomeres 5 and 6^11,12 (R5/R6), we show spatial control of plasmid transfection in NM and NL. In addition, we show temporal regulation of expression 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)⁸.

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 ensures proper embryo positioning for electroporation and optimal viability, respectively.
   NOTE: After 48-52 h of incubation, the eggs will be at Hamburger-Hamilton (HH) stage ~12-13 and ready for electroporation.

2. Preparation

1. Plasmid Mixing
   1. Add 1 µL of 0.1% fast green (0.1% in 18 MΩ deionized and distilled H₂O [ddH₂O]) for every 7 µL of DNA mixture.
   2. For co-electroporation of multiple plasmids, mix plasmids in a 1:1 ratio. The final DNA concentration should be 5-8 µg/µL.
2. Fill Injection Pipette
   1. Pull pipettes on a micropipette puller. Fill the pipette with 1-2 µL plasmids using a 28G syringe needle.
   2. Break pipette tip to 10 - 20 µm using forceps. Use a microscope to assist in determining the size of the broken pipette tip. Attach the pipette to the pipette holder of the picospritzer.

3. Windowing

NOTE: The windowing procedure has been published before¹³. Please see reference 13 for additional visual information.

1. Wipe all instruments and the work area with 70% ethanol.
2. Take the desired number of set eggs out of the incubator (between 6-10), leave at room temperature, and individually swab eggs with 70% ethanol again. Eggs remain viable for at least 2 h after incubator removal.
3. Place the egg on top of a light illuminator to identify embryo positioning. Circle the center of the dark area (i.e., embryo) with a pencil.
4. Using a 19G needle, poke a hole in the blunt end of the egg.
5. Poke another hole near the drawn circle on the pointed side of the egg. Remove a small piece of the outer eggshell (about 16 mm²) with the 19G needle and then remove a small piece of the inner eggshell (about 8 mm²) with forceps.
6. With a 3 mL syringe fitted with a 19G needle, withdraw 1.5-2.5 mL of albumin from the egg through the blunt-end hole. Be sure to angle the needle down at 45°.
7. Cover the blunt-end hole with a small piece of tape (1 cm x 1 cm). Cover the drawn circle and the second hole with tape (2.5 cm x 4 cm).
8. With curved scissors (cutting edge = 2.5 cm), cut a window within the circle. Scissors should be parallel to the eggshell and the diameter of the window should be less than 2 cm.

4. Plasmid Injection

1. Turn on the picospritzer. Set the pressure to 18 psi and the duration to 5 µs. Turn on the air tank and lamp for the dissection microscope.
2. Make 30 mL stock solution of a 1:10 dilution of store-bought Indian ink mixed in sterile phosphate-buffered saline (PBS). Store at 4 °C. Injection of Indian ink is an important step to get a better visualization of the chicken embryo.
   1. Fill a 1 mL syringe with the diluted Indian ink solution. Attach a 27G needle and expel any bubbles that are present in the syringe.
   2. Insert needle just under the yolk membrane ~2 - 3 mm from the embryo and inject ~0.2 mL of Indian ink gently under the embryo. Do not inject more than 0.5 mL of the Indian ink as it may decrease embryo survival.
3. Place the windowed egg on an egg holder under the dissection microscope. Use the highest magnification for best visualization of plasmid injection site.
4. Using forceps, remove the membrane over the injection area. Auditory brainstem regions of interest (i.e., NM and NL) arise from Rhombomeres 5 and 6 (R5/R6). R5/R6 are located adjacent to the otocysts (an embryonic structure in vertebrates that develops into the inner ear) that serve as landmarks for R5/R6 plasmid injections.
5. Lower the DNA-filled pipette into the neural tube overlying the R5/R6 region and between otocysts using the micromanipulator. A schematic of ideal placement is shown in Figure 1A.
6. Apply air pressure (18 psi, 5 µs duration) from the picospritzer to eject DNA in the neural tube.

5. Electroporation

1. Turn on the current/voltage stimulator.
2. Fill a 3 mL syringe with PBS and attach the syringe filter. Add 1-2 drops of PBS onto the embryo.
3. Lower the bipolar electrode to the embryo using the micromanipulator with the negative electrode above the injection site (medial) and the positive electrode lateral to the R5/R6 (Figure 1A). Avoid direct contact with the embryo. Use bipolar electrodes where the electrode material is platinum iridium. Adjust the spacing between the tips to 0.5 mm with forceps.
4. Using a current/voltage stimulator, pulse 20 times at 50 V for 1 ms at 1 s intervals.
5. After electroporation, add one drop of PBS over the exposed area. Gently remove the electrode and clean it with a laboratory tissue and 70% ethanol. Close the window exposing the embryo with tape.
6. Label eggshell with the injected plasmid type and the hatch date.
7. Place eggs back into incubator with window side up. Incubate at 38 °C with 50% humidity until desired developmental stage is reached.
8. If a tet-on vector is electroporated for temporal control of gene expression, apply Doxycycline (Dox) every 24 h to induce gene expression (see Section 6).

6. Tet-on System for Temporal Control of Gene Expression

1. Use the following plasmids:
   pCAGGS-T2TP: a transposase expressing plasmid.
   pT2K-CAGGS-rtTA-M2: a plasmid that stably expresses the Dox-binding protein.
   pT2K-BI-TRE-EGFP: a plasmid that contains a bidirectional tet-on promoter (TRE)-driving expression of the EGFP reporter and a second gene of interest.
   NOTE: The above three plasmids need to be co-electroporated in a 1:1:1 ratio.
2. Stock solution preparation:
   1. Dissolve Dox in sterile PBS to produce a 1 mg/mL stock solution. Store at -20 °C.
3. Dox application:
   1. To induce the expression of the gene of interest during certain developmental stages, apply Dox every 24 h.
   2. When applying Dox, take the egg out, open the window, pipette 50 µL Dox onto the chorioallointoic membrane, close the window and put the egg back into the incubator.

7. Preparation of Dissecting Area for Brainstem Slices

NOTE: The following sections (7 - 9) and procedures have been published before¹⁴. Please see these references for additional visual information.

1. Prepare an agar solution (40 mg/mL agarose, i.e. 4% in ddH₂O). Pour agar solution into a Petri dish and allow it to solidify at room temperature. Store covered agar plate in the refrigerator for future use during brainstem slicing of tissue.
2. Continually bubble artificial cerebral spinal fluid (ACSF) with 95% O₂ and 5% CO₂ (pH 7.2-7.4 and osmolarity: 295-310 mOsm/L).
3. Clean the working area and vibratome with 70% EtOH and rinse the blade with distilled water.
4. Place a clean fluid absorption pad on the working area with appropriate dissecting tools.
5. Take the agar plate out of the refrigerator, cut a small agar cube (10 mm x 10 mm x 8 mm). Glue the agar block to the stage of a vibratome-slicing chamber using commercially available superglue.

8. Isolation of Chicken Auditory Brainstem

1. Open the egg at taped window site. Puncture the membrane sac with a scalpel and remove the head of the chicken embryo.
2. Decapitate the chicken with sharp scissors.
3. Place the razor blade tip slightly posterior to the eyes. Incise through the skull at the rostral-to-caudal midline. Apply slight pressure depending on the age of the embryo. Older embryos require more pressure.
4. Gently push aside skin and feathers to expose skull and verify midline cut.
5. Applying strong pressure, slice the rostral portion of skull with a razor blade. Immediately place blade posterior to eyes and cut through entire skull and brain tissue.
6. Make midline-to-lateral incisions with scissors in the skull"s caudal region, slightly anterior to neck muscles on both sides of the head. Expose brain and cerebellum by pulling away skull and excess tissue.
7. Cut tissue attached to the brainstem with scissors and remove the brainstem, which freely detaches from the skull.

9. Preparation of Brainstem Slices for In Vivo Electrophysiology or Imaging

1. Pin down brainstem through the optic tecta.
2. Remove the cerebellum by cutting peduncles with scissors, exposing the floor of the fourth ventricle.
3. Using tweezers, remove any membranous tissue and blood vessels from the brainstem surface.
4. To block the brainstem, make a horizontal cut at the most rostral end of the floor of the fourth ventricle, just caudal to the cortex. Make lateral cuts perpendicular through the optic tecta to isolate brainstem.
5. Place a small amount of super glue on the vibratome stage directly in front of the agar block.
6. Lift the brainstem at the spinal cord using tweezers and place it on the super glue with rostral side down and dorsal side towards the vibratome blade. Remove excess glue with a laboratory tissue or filter paper.
7. Pour oxygenated ACSF into the vibratome stage. Set the vibratome blade at a 20-22° angle. Start vibratome at maximum-amplitude oscillation. Move the stage up towards the blade so the top of the tissue is parallel with the blade.
8. Rapidly move blade towards brainstem tissue. Slow down the blade considerably prior to the blade"s contact with tissue.
9. Slice tissue with the slowest possible forward speed. Once the blade is through the entire coronal tissue section, gently remove the slice using a transfer pipette.
10. Lower the stage 200-300 µm and slice again. Repeat until anatomical landmarks of auditory nuclei become visible, e.g., the distinct dorsal/ventral neuropil region of NL and the medial location of NM relative to NL.
11. Carefully maintain slices in ACFS. This can be done at room temperature (22° C) or near physiological conditions (~41° C) using a warm water bath. Note the order of slicing. The first slice corresponds to the most caudal region of tissue and the last slice corresponds to the most rostral region. This roughly represents the tonotopic organization of the auditory brainstem, from low- to high-frequency encoding regions, respectively.

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

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		37.8° 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