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).

1. Ventral Laparotomy
<ol>
 <li> 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 &mu;L per gram body weight). Working anesthetic solution: 180 &mu;L of 50 mg/mL pentobarbital sodium solution; 100 &mu;L of absolute ethanol; 320 &mu;L of 65 mg/mL aqueous magnesium sulfate (modulates uterine tone); and 400 &mu;L of propylene glycol (vehicle miscibl...

<ol>
	<li>Gillespie, P. G., Muller, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanotransduction+by+hair+cells:+models,+molecules,+and+mechanisms.">Mechanotransduction by hair cells: models, molecules, and mechanisms.</a> Cell. 139, 33-44 (2009).</li><li>Bok, J., Chang, W., Wu, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+and+morphogenesis+of+the+vertebrate+inner+ear.">Patterning and morphogenesis of the vertebrate inner ear.</a> Int. J. Dev. Biol. 51, 521-533 (2007).</li><li>Kelley, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cell+fate+in+the+sensory+epithelia+of+the+inner+ear.">Regulation of cell fate in the sensory epithelia of the inner ear.</a> Nat. Rev. Neurosci. 7, 837-849 (2006).</li><li>Ohyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+signaling+is+necessary+for+patterning+the+sensory+and+nonsensory+regions+of+the+developing+mammalian+cochlea.">BMP signaling is necessary for patterning the sensory and nonsensory regions of the developing mammalian cochlea.</a> J. Neurosci. 30, 15044-15051 (2010).</li><li>Pan, W., Jin, Y., Stanger, B., Kiernan, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+is+required+for+the+generation+of+hair+cells+and+supporting+cells+in+the+mammalian+inner+ear.">Notch signaling is required for the generation of hair cells and supporting cells in the mammalian inner ear.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 15798-15803 (2010).</li><li>Gubbels, S. P., Woessner, D. W., Mitchell, J. C., Ricci, A. J., Brigande, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+auditory+hair+cells+produced+in+the+mammalian+cochlea+by+in+utero+gene+transfer.">Functional auditory hair cells produced in the mammalian cochlea by in utero gene transfer.</a> Nature. 455, 537-541 (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide for the Care and Use of Laboratory Animals. , (2010).</li><li>Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+expression+of+transgenes+introduced+by+in+vivo+electroporation.">Controlled expression of transgenes introduced by in vivo electroporation.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 1027-1032 (2007).</li><li>Chen, C., Smye, S. W., Robinson, M. P., Evans, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+electroporation+theories:+a+review.">Membrane electroporation theories: a review.</a> Med. Biol. Eng. Comput. 44, 5-14 (2006).</li><li>Saito, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electroporation+in+the+embryonic+mouse+central+nervous+system.">In vivo electroporation in the embryonic mouse central nervous system.</a> Nat. Protoc. 1, 1552-1558 (2006).</li><li>Brigande, J. V., Gubbels, S. P., Woessner, D. W., Jungwirth, J. J., Bresee, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation-mediated+gene+transfer+to+the+developing+mouse+inner+ear.">Electroporation-mediated gene transfer to the developing mouse inner ear.</a> Methods Mol. Biol. 493, 125-139 (2009).</li><li>Morsli, H., Choo, D., Ryan, A., Johnson, R., Wu, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+mouse+inner+ear+and+origin+of+its+sensory+organs.">Development of the mouse inner ear and origin of its sensory organs.</a> J. Neurosci. 18, 3327-3335 (1998).</li><li>Sher, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+embryonic+and+postnatal+development+of+the+inner+ear+of+the+mouse.">The embryonic and postnatal development of the inner ear of the mouse.</a> Acta. Otolaryngol. , 1-77 (1971).</li><li>Sheffield, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+vector+tropism+for+supporting+cells+in+the+developing+murine+cochlea.">Viral vector tropism for supporting cells in the developing murine cochlea.</a> Hear Res. 277, 28-36 (2011).</li><li>Bedrosian, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+delivery+of+recombinant+viruses+to+the+fetal+murine+cochlea:+transduction+characteristics+and+long-term+effects+on+auditory+function.">In vivo delivery of recombinant viruses to the fetal murine cochlea: transduction characteristics and long-term effects on auditory function.</a> Mol. Ther. 14, 328-335 (2006).</li><li>Reisinger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+functional+equivalence+of+otoferlin+and+synaptotagmin+1+in+exocytosis.">Probing the functional equivalence of otoferlin and synaptotagmin 1 in exocytosis.</a> J. Neurosci. 31, 4886-4895 (2011).</li><li>Magnani, E., Bartling, L., Hake, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Gateway+to+MultiSite+Gateway+in+one+recombination+event.">From Gateway to MultiSite Gateway in one recombination event.</a> BMC Mol. Biol. 7, 46 (2006).</li><li>Perret-Gentil, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Veterinary Suturing. , .</li><li>Oesterle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> P-1000 &#38; P-97 Pipette Cookbook. , (2011).</li></ol>

Gene transfer to the developing mouse inner ear: The mouse inner ear develops from the otic placode during the first week of postimplantation development12,13. 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...

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

gene transfer to the developing mouse inner ear by in vivo electroporation

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.

Watch this Scientific Journal Video about Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation at JoVE.com

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

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.

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

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 ...

gene-transfer-to-developing-mouse-inner-ear-vivo

Research

JoVE Journal

Neuroscience

13.9K visualizações.  Oregon Health & Science University. A orelha interna do rato é um órgão sensorial placode derivado cujo programa é elaborado de desenvolvimento durante a gestação. Nós definimos uma In utero Técnica de transferência de gene consiste de três etapas: mouse laparotomia ventral, microinjeção transuterina, e In vivo Eletroporação. Nós usamos a microscopia de vídeo digital para demonstrar as críticas técnicas experimentais embriológicos.