Gene Expression > Detection in Cells and Tissues

User Ratings

Your rating: None
Your rating: None
Your rating: None
 Add your comments

Transfection of Mouse Cochlear Explants by Electroporation

Elizabeth C. Driver1,  Matthew W. Kelley1

1National 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
GO TO THE FULL TEXT:
PDF or HTML at Wiley Online Library
Are you the author of this protocol? Login or register and return to this page.

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

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

 Basic Protocol
 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, 1998) and euthanizing the mouse (Donovan and Brown, 2006)
Looking for materials?  Suppliers Guide
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  • Figure 4.34.1
    Isolation of developing bony labyrinth of the inner ear. (A) Dorsal view of the head of a mouse at E14.5. Dotted line indicates dorsal midline. The skull should be opened along this line followed by removal of the brain. (B) Once the brain has been removed, the developing bony labyrinth of the inner ear (outlined) can be visualized in the temporal bone located in the ventral floor of the skull (arrow). The bony labyrinth can be isolated by dissecting around its borders (indicated by arrowheads). (C) Ventral view of the isolated bony labyrinths. Cochlear (C) and vestibular (V) regions are indicated. (D) Anterior view of the bony labyrinths oriented as in C, illustrating the natural curvature between the cochlear (C) and vestibular (V) regions.

    View Image
  • Figure 4.34.2
    Isolation of the developing cochlear duct and sensory epithelium. (A) The bony labyrinth should be oriented with the ventral side up and immobilized by placing two minutien pins through the vestibular region. Once immobilized, it will be possible to identify the base of the cochlear duct through the bony labyrinth. The line drawing on the right illustrates the shape of the cochlear spiral. (B) Beginning in the base, use fine forceps to make an opening (straight arrow) and extend it parallel to the duct (curved arrow). (C) Use forceps to continue to increase the size of the opening in the bony labyrinth by working along the outside edge of the cochlea (arrow). (D) Once the ventral surface of the bony labyrinth of the cochlea has been removed, the developing cochlear duct can be visualized (arrows). (E) To expose the developing sensory epithelium of the cochlea, carefully remove the upper (ventral) half of the duct using fine forceps (arrow). Following removal of the upper half of the duct, the remainder of the bony labyrinth of the cochlea can also be removed. (F) At this point, the developing sensory epithelium (organ of Corti) is completely exposed (arrow). Next, separate the cochlea from the vestibular region of the ear by using fine forceps to cut along the dotted line. (G) Ventral view of the isolated cochlear spiral with basal and apical ends indicated. (H) In a side view, the epithelium is present as a spiral that extends from the base to the apex (arrows). The lower region of the cochlea is comprised of mesenchymal derivatives (note small blood islands, arrowheads) and developing spiral ganglion neurons. (I) For electroporation, the cochlea should be oriented between the electrodes with the base located closer to the negative electrode (indicated in the image). (J) Schematic diagram depicting the main dissection steps: isolation of the inner ear, removal of overlying cartilage, opening the cochlear duct, and isolation of the cochlear sensory epithelium.

    View Image
  • Figure 4.34.3
    Examples of cochlear electroporations. (A) Low-magnification image of an explant established on E13.5 and maintained for 6 days in vitro. Hair cells are labeled with an antibody against Myosin VI (red) while transfected cells are labeled with anti-GFP (green). Apex and base of the cochlea are indicated. (B) A higher magnification view of a transfected explant treated as in A. Transfected cells are present in Kölliker's organ (KO), the sensory epithelium (SE), and in the lesser epithelial ridge (LER). (C) High-magnification view of cells transfected with an Atoh1 expression vector (green). Endogenous hair cells within the SE are labeled with anti-Myosin VI (red). Atoh1-transfected cells located in the SE (arrow) or in KO (arrowheads) have also developed as hair cells. Note that virtually all transfected cells appear yellow as a result of expression of the hair cell marker Myosin VI. (D) High magnification view of Atoh1-transfected cells within the sensory epithelium. Each transfected cell has developed as a hair cell. (E) Cluster of Atoh1-transfected cells located in KO. Actin is labeled in blue. The induction of a group of hair cells leads to an accumulation of actin that is similar to what is observed in the SE. (F) Cells transfected with the inhibitory bHLH, Id3 (green), are predominantly inhibited from developing as hair cells.

    View Image

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.
    Holt, J.R. 2002. Viral-mediated gene transfer to study the molecular physiology of the Mammalian inner ear. Audiol. Neurootol. 7:157-160.
    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.
    Kelley, M.W. 2006. Regulation of cell fate in the sensory epithelia of the inner ear. Nat. Rev. Neurosci. 7:837-849.
    Lopez, A., Rols, M.P., and Teissie, J. 1988. 31P NMR analysis of membrane phospholipid organization in viable, reversibly electropermeabilized Chinese hamster ovary cells. Biochemistry 27:1222-1228.
    Luebke, A.E., Foster, P.K., Muller, C.D., and Peel, A.L. 2001. Cochlear function and transgene expression in the guinea pig cochlea, using adenovirus- and adeno-associated virus-directed gene transfer. Hum. Gene Ther. 12:773-781.
    Mir, L.M. 2008. Application of electroporation gene therapy: Past, current, and future. Methods Mol. Biol. 423:3-17.
    Neumann, E., Schaefer-Ridder, M., Wang, Y., and Hofschneider, P.H. 1982. Gene transfer into mouse lyoma cells by electroporation in high electric fields. Embo. J. 1:841-845.
    Rocha, A., Ruiz, S., and Coll, J.M. 2002. Improvement of DNA transfection with cationic liposomes. J. Physiol. Biochem. 58:45-56.
    Rols, M.P. 2008. Mechanism by which electroporation mediates DNA migration and entry into cells and targeted tissues. Methods Mol. Biol. 423:19-33.
    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.
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library
Looking for Answers?
Do you have tips, tricks, or improvements to share?

Join the Conversation

Post new comment

The content of this field is kept private and will not be shown publicly.
CAPTCHA
This question is for testing whether you are a human visitor and to prevent automated spam submissions.