Transfection of Mouse Cochlear Explants by Electroporation

Elizabeth C. Driver1, Matthew W. Kelley1

1 National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 4.34
DOI:  10.1002/0471142301.ns0434s51
Online Posting Date:  April, 2010
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Abstract

The sensory epithelium of the mammalian inner ear, also referred to as the organ of Corti, is a remarkable structure comprised of highly ordered rows of mechanosensory hair cells and non‐sensory supporting cells located within the coiled cochlea. This unit describes an in vitro explant culture technique that can be coupled with gene transfer via electroporation to study the effects of altering gene expression during development of the organ of Corti. While the protocol is largely focused on embryonic cochlea, the same basic protocol can be used on cochleae from mice as old as P5. Curr. Protoc. Neurosci. 51:4.34.1‐4.34.10. © 2010 by John Wiley & Sons, Inc.

Keywords: hair cell; hearing; cell fate; mechanosensory cells; organ of Corti

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • DMEM (Invitrogen, cat. no. 12430)
  • Matrigel (BD Biosciences)
  • Pregnant mouse at desired gestational stage
  • 70% (w/v) ethanol
  • HBSS/HEPES, cold (see recipe)
  • Sylgard‐charcoal‐coated glass petri dishes, 60 mm and 100 mm (see recipe)
  • Plasmid DNA, expression vector of choice, at 1.5 mg/ml in water (details of plasmid selection criteria are included in the Commentary)
  • Cochlear explant culture medium (see recipe)
  • OS‐30 solvent (Dow‐Corning)
  • Appropriate antibody (e.g., anti‐MyosinVI or anti‐Myosin VIIa)
  • 15‐ml conical tubes
  • 60‐ or 100‐mm tissue culture dishes
  • 35‐mm tissue culture dishes, No. 0 coverslips (MatTek)
  • 37°C incubator
  • Dissection instruments including:
    • Scissors
    • No. 5 forceps
    • Dissecting microscope
  • Minutien pins (Fine Science Tools)
  • Electroporation equipment including:
    • Electrodes
    • Electroporator (ECM‐830, BTX)
  • Additional reagents and equipment for anesthetizing (Donovan and Brown, ) and euthanizing the mouse (Donovan and Brown, )
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Figures

Videos

Literature Cited

Literature Cited
   Anwer, K. 2008. Formulations for DNA delivery via electroporation in vivo. Methods Mol. Biol. 423:77‐89.
   Chernomordik, L.V., Sokolov, A.V., and Budker, V.G. 1990. Electrostimulated uptake of DNA by liposomes. Biochim. Biophys. Acta 1024:179‐183.
   Dabdoub, A., Puligilla, C., Jones, J.M., Fritzsch, B., Cheah, K.S., Pevny, L.H., and Kelley, M.W. 2008. Sox2 signaling in prosensory domain specification and subsequent hair cell differentiation in the developing cochlea. Proc. Natl. Acad. Sci. U.S.A. 105:18396‐18401.
   Di Pasquale, G., Rzadzinska, A., Schneider, M.E., Bossis, I., Chiorini, J.A., and Kachar, B. 2005. A novel bovine virus efficiently transduces inner ear neuroepithelial cells. Mol. Ther. 11:849‐855.
   Donovan, J. and Brown, P. 1998. Anesthesia. Curr. Protoc. Immunol. 27:1.4.1‐1.4.5.
   Donovan, J. and Brown, P. 2006. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
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   Jones, J.M., Montcouquiol, M., Dabdoub, A., Woods, C., and Kelley, M.W. 2006. Inhibitors of differentiation and DNA binding (Ids) regulate Math1 and hair cell formation during the development of the organ of Corti. J. Neurosci. 26:550‐558.
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   Sobkowicz, H.M., Bereman, B., and Rose, J.E. 1975. Organotypic development of the organ of Corti in culture. J. Neurocytol. 4:543‐572.
   Stone, I.M., Lurie, D.I., Kelley, M.W., and Poulsen, D.J. 2005. Adeno‐associated virus‐mediated gene transfer to hair cells and support cells of the murine cochlea. Mol. Ther. 11:843‐848.
   Stulen, G. 1981. Electric field effects on lipid membrane structure. Biochim. Biophys. Acta 640:621‐627.
   Woods, C., Montcouquiol, M., and Kelley, M.W. 2004. Math1 regulates development of the sensory epithelium in the mammalian cochlea. Nat. Neurosci. 7:1310‐1318.
   Zheng, J.L. and Gao, W.Q. 2000. Overexpression of Math1 induces robust production of extra hair cells in postnatal rat inner ears. Nat. Neurosci. 3:580‐586.
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