Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

Summary

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

Abstract

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing ¹. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth ^2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes ^4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development⁶.

The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos⁷. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells^8-10.

We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol¹¹. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Protocol

1. Ventral Laparotomy

1. Anesthetize a dam whose embryos are at embryonic day 11.5 (E11.5; noon on the day a vaginal plug is detected is day 0.5 of embryonic development) by intraperitoneal injection of sodium pentobarbital anesthetic solution (7.5 μL per gram body weight). Working anesthetic solution: 180 μL of 50 mg/mL pentobarbital sodium solution; 100 μL of absolute ethanol; 320 μL of 65 mg/mL aqueous magnesium sulfate (modulates uterine tone); and 400 μL of propylene glycol (vehicle miscible with aqueous and organic components).
2. Assess completeness of anesthesia by conducting noxious stimuli tests: paw squeeze; tail pinch; and blink response to cheek and vibrissae touch. Apply sterile ophthalmic ointment to the corneas.
3. Shave the abdominal fur from the suprapubic area to the rib cage with shears and a fine blade (Oster #40 blade). Disinfect the abdomen with 70% ethanol, 10% povidone iodine (Betadine), and 70% ethanol, sequentially. Place mouse abdomen-side down on a sterile drape and then set on a heating pad or warm plate (37 °C) for 2-5 mins.
4. Incise the abdominal skin in the ventral midline with ball-tipped scissors. Extend the incision for 10-14 mm. Identify the linea alba, an avascular, white connective tissue band along the ventral midline of the abdominal wall. Incise the linea alba with ball tipped scissors and extend the incision 10-14 mm. Immediately irrigate the abdomen with prewarmed (37 °C) lactated Ringer's solution.
5. Externalize the two horns of the uterus with ring forceps without applying excessive pressure on the implantation sites. Irrigate the externalized uterine horns with prewarmed, lactated Ringer's solution.

2. Transuterine Microinjection

1. Fabricate a thick-walled, borosilicate glass capillary microinjection pipette with the following characteristics: 12-16 μm outer diameter and a 20 degree bevel. On a Sutter P-97 pipette puller with small box filament, use the following program: heat = ramp test plus 3 units; pressure = 200; pull = 0; velocity = 46; time = 100). With the Sutter BV-10 beveler, use the 104C (gold) abrasive disk for large diameter pipettes. Resuspend expression plasmid at 3-4 μg/μL in calcium free phosphate buffered saline (pH 7.2-7.4). Add crystalline fast green to the concentrated DNA, vortex gently for 30 seconds, and spin at 10,000 g for 10 seconds. The minimum amount of fast green required to visually track the efficacy of the injection is determined empirically. Backfill the beveled pipette with the DNA/fast green solution. Connect the loaded microinjection pipette to the pipette holder of the pressure injector (Picospritzer using >99% pure nitrogen as source gas).
2. Transilluminate the uterus with low intensity, halogen light to visualize the embryo within its implantation site. Identify the beating heart, brain vesicles, limb buds, nascent 4^th ventricle of the hindbrain, and eye. Irrigate the uterus every 2 minutes with prewarmed, lactated Ringer's solution to maintain hydration. Apply gentle pressure on the uterus to reorient the embryo and identify the anatomical landmarks noted above.
3. Orient the embryo to identify the primary head vein whose anterior and posterior branches flank the mesenchymal territory in which the otocyst resides. The otocyst proper cannot be seen by transillumination of the uterus. The otocyst is located midway between the anterior and posterior branches of the primary head vein which, along with the main trunk of the vein, form the shape of the uprights or goalposts on an American football field. The otocyst is midway between the uprights.
4. Insert the injection pipette through the uterus in a trajectory in line with the presumptive location of the otocyst. Pulse the microinjector once after passing through the uterus to visualize the tracer dye and the approximate the location of the pipette tip. Advance the pipette under micrometer control and pulse again to assess depth. Further advance the pipette into the lateral head mesenchyme and pulse repeatedly. Successful otocyst targeting will reveal the tapered shape of the endolymphatic duct dorsally and the scallop shell-shape of the vestibule. Release pressure on the uterus, remove the pipette from the embryo/uterus in one motion, and immediately irrigate the uterus with prewarmed, lactated Ringer's solution.

3. In vivo Electroporation

1. Irrigate the uterus with lactated Ringer's solution. Freshly applied lactated Ringer's solution is necessary to electrically couple the paddle-style electrodes to the uterus. Moisten the tungsten surfaces of the electrodes with lactated Ringer's. Center the injected otocyst in the path of the electrodes. Gently compress the uterus with the electroporation paddles. The cathode is in contact with the uterine wall lateral to the injected otocyst and the anode is in contact with the uterine wall adjacent to the uninjected otocyst. Trigger a square wave pulse train with the foot pedal switch on the electroporator. Electroporation parameters are: 5, 50 msec pulses at 43 volts per pulse and a 950 msec interpulse delay. Immediately irrigate the uterus after the pulse train is delivered. Record the current delivered to the tissue: 60-100 mAmps is sufficient to transfect otic epithelial progenitors. Inject and electroporate 4-6, E11.5 embryos per dam.
2. Perform a second, independent transuterine microinjection of aqueous fluorescent dextran (Alexa Fluor 488 if the expression plasmid encodes a red fluorescent protein or Alex Fluor 594 if the expression plasmid encodes a green fluorescent protein) into the 4^th ventricle of those embryos whose inner ears are accurately injected and at least 60 mAmps of current per pulse was delivered during electroporation. The fluorescent dextran will be detectable at birth in the hindbrain and enable selection of pups whose inner ears were manipulated during embryogenesis (see 3.5).
3. Irrigate the uterine horns with lactated Ringer's. Reinsert the uterine horns into the abdominal cavity. Flush the uterine cavity with 2-4 mL of prewarmed, lactated Ringer's solution and allow the overflow to drain out of the incision site onto the sterile drape. Replace the draping with dry, sterile material. Suture the abdominal wall with a non-cutting needle and a 6-0 resorbable suture. We prefer a running stitch, locking every other stitch, for both the abdominal wall and skin.
4. Dry the dams' fur and administer a non-steroidal anti-inflammatory such as Meloxicam by subcutaneous injection. Return the dam to the prewarmed recovery cage on a sterile drape. Monitor and record the dams' respirations, incision site patency, and vagina for bloody discharge. Bleeding is rare but if present and unabated, euthanize the dam while she is still under anesthesia. Note the time she regains consciousness and attempts to ambulate. Return the dam to the mouse colony when she shows signs of eating and drinking and has begun to nest build. Typically, this occurs within 12 hrs.
5. At birth (postnatal day 0), flash the hindbrain region of each pup in the litter to detect the fluorescent dextran using a stereofluorescence dissecting microscope with a GFP or Texas Red filter set as appropriate. Return to the lactating dam only those pups that display hindbrain labeling.

4. Representative Results

Figure 1. Electroporation-mediated gene transfer to the developing cochlea. The E11.5 otocyst was injected with an expression plasmid encoding enhanced green fluorescent protein (EGFP) and electroporated (pulse train parameters: five, 43 volt pulses at 50 msec/pulse and 950 msec interpulse delay). A) A representative inner ear from a postnatal day 6 (P6) pup whose otocyst was injected and electroporated at E11.5 demonstrating EGFP expression from the base through the middle turn of the cochlea. The lateral wall of the cochlea was removed from the middle turn and apex only. E11.5 progenitors that give rise to the apex were not transfected. B) Whole mount immunostaining of the cochlea in (A) with the hair cell marker, myosin 7a (Myo7a), indicates that EGFP expression follows the trajectory of the organ of Corti and is grossly localized to the hair cell-bearing sensory epithelium. C) A representative inner ear from an E18.5 embryo whose otocyst was injected and electroporated at E11.5. The laser confocal projection demonstrates EGFP expression in Myo7a-positive sensory hair cells. D) Laser confocal projection of the cochlear sensory epithelium from the E18.5 organ of Corti indicating EGFP expression in the inner hair cells (ihc), outer hair cells (ohc), inner phalangial cells (ipc), pillar cells (pc), and Deiters' cells (dc). The scale bar in (B) applies to (A).

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Discussion

Gene transfer to the developing mouse inner ear: The mouse inner ear develops from the otic placode during the first week of postimplantation development^12,13. By embryonic day 9.5 (E9.5), the placode has invaginated and morphed into a fluid-filled vesicle called the otocyst². Otic precursors in the vesicle give rise to the sensory and nonsensory cells within the mature inner ear as well as the neurons that innervate mechanically sensitive hair cells in the vestibular and auditory s...

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Materials

Name	Company	Catalog Number	Comments
Micro Sterilizing Case	Roboz Surgical Instruments Co.	RS-9900a	8X8.5X1.25 inches
Ball-tipped scissors	Fine Science Tools	14109-09
Ring forceps	Fine Science Tools	11106-09	4.8mm ID/6mm OD
Adson Tissue Forceps	Fine Science Tools	11027-12
Needle driver	Fine Science Tools	12502-12
Allergy Syringe Tray	BD Biosciences	305536
Suture 6-0	Syneture	GL-889	0.7 metric gastrointestinal suture
Lactated Ringer’s Injection USP	Baxter Internationl Inc.	2B2323
Fast green	Sigma-Aldrich	F7258
Borosilicate glass capillary	Harvard Apparatus	30-0053
Nembutal Sodium Solution	OVATION Pharmaceuticals	NDC 67386-501-52
MgSO4.7H2O	Fisher Scientific	M63-500
Propylene glycol	Fisher Scientific	P355-1
Ethanol	Sigma-Aldrich	E7023-500
Meloxicam	Boehringer Ingeheim	NADA 141-219
Micropipette Puller	Sutter Instrument Co.	P-97	FB255B box filament; consult Pipette Cookbook from Sutter instruments
Micr–lectrode Beveler	Sutter Instrument Co.	BV-10	104C beveling disk for large pipettes; consult owner’s manual for beveling theory
Micropipette holder	Warner Instruments	MP-S15T	For 1.5mm outer diameter pipette and female pressure port for Picospritzer tubing.
Tweezers-style electrode	Protech International, Inc.	CUY650P5	5 mm outer diameter
Square Wave Electroporator	Protech International, Inc.	CUY21EDIT	Footpedal recommended
PICOSPRITZER III	Parker Hannifin Corporation	051-0500-900	Footpedal recommended
Manual Control Micromanipulator	Harvard Apparatus	640056
Horizontal laminar flow clean bench	Envirco		Custom modifications to LF 630-10554. See supplementary information for hood schematic.
Leica stereofluorescence dissecting microcope with Lumencor SOLA light engine	Bartels and Stout and Lumencor	MZ10F with Lumencor SOLA light engine	Footpedals to focus the MZ10F and to trigger the SOLA light engine are recommended
Alexa Fluor 594 Dextran	Invitrogen	D22913	10mg/ml, aqueous
Alexa Fluor 488 Dextran	Invitrogen	D22910	10mg/ml, aqueous
Enviro-dri	Shepherd Specialty Papers		www.ssponline.com