Перенос генов в разработке мыши Внутреннее ухо по<em> В Vivo</em> Электропорация
Summary
Мышь внутреннее ухо плакоды производных органа чувств чье развитие программа разработана во время беременности. Мы определяем<em> Внутриутробно</em> Перенос генов техника, состоящая из трех этапов: мышь вентральной лапаротомия, transuterine микроинъекции, и<em> В естественных условиях</em> Электропорации. Мы используем цифровой микроскопии видео, чтобы продемонстрировать критической экспериментальных эмбриологических методов.
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 cells8-10.
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
Discussion
Перенос генов в развивающихся мыши внутреннего уха: мышь внутреннее ухо развивается из слуховой плакоды в течение первой недели постимплантационных развитии 12,13. В эмбриональном день 9,5 (E9.5), плакоды был инвагинировать и превратился в заполненных жидкостью пузырьков на?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Humana Press for permission to publish the microinjection pipette fabrication figure which originally appeared on page 130 of reference 11; Larry Dlugas and Steven Wong, OHSU Department of Educational Communications, for videography; Larry Dlugas for video design and editing; Adam M. O’Quinn, Senior Designer, Trion/Envirco for designing our custom horizontal laminar flow hood and Les Goldsmith for providing the technical schematic; Victor Monterroso, MV, MS, PhD and Tom Chatkupt, DVM, OHSU Department of Comparative Medicine, for guidance with our animal care protocol, surgical techniques, and prophylactic analgesia regimen; Marcel Perret-Gentil, DVM, MS, for sharing his handout on veterinary suturing techniques; Edward Porsov, MS, for designing our Adobe Premiere Pro video microscopy computer workstation; and Leah White and Jonas Hinckley of LNS Captioning (Portland, OR). This work was supported by grants from the National Institute on Deafness and other Communication Disorders: DC R01 008595 and DC R01 008595-04S2 (to JB) and P30 DC005983 (Oregon Hearing Research Center Core Grant, Peter Gillespie, Principal Investigator).
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Micro Sterilizing Case | ROBOZ | 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 | Becton Dickison | 305536 | |
| Suture 6-0 | Syneture | GL-889 | 0.7 metric gastrointestinal suture |
| Lactated Ringer’s Injection USP | Baxter | 2B2323 | |
| Fast green | Sigma Aldrich | F7258 | |
| Borosilicate glass capillary | Harvard Apparatus | 30-0053 | |
| Nembutal Sodium Solution | OVATION Pharmaceuticals Inc. | 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 Instruments | P-97 | FB255B box filament; consult Pipette Cookbook from Sutter instruments |
| Microelectrode Beveler | Sutter Instruments | 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 | 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 |
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