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

摘要

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.

摘要

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.

方案

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 res....

讨论

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.......

材料

本文使用的材料清单

姓名	公司	目录编号	评论
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