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

Lingyan Wang, Han Jiang, John Brigande

Research output: Contribution to journalArticle

13 Citations (Scopus)

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 development 6. 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 embryos 7. 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 protocol 11. 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.

Original languageEnglish (US)
Article numbere3653
Pages (from-to)1
Number of pages1
JournalJournal of Visualized Experiments
Issue number64
DOIs
StatePublished - Jun 30 2012

Fingerprint

Gene transfer
Electroporation
Inner Ear
Embryonic Structures
Canals
Genes
Plasmids
Organ of Corti
Cells
Semicircular Canals
Gene Transfer Techniques
Cochlea
Microinjections
Audition
Laparotomy
Auditory Hair Cells
Epithelium
Vestibular Hair Cells
Labels
Transducers

Keywords

  • Developmental biology
  • Genetics
  • In vivo electroporation
  • Inner ear
  • Issue 64
  • Neuroscience
  • Otocyst
  • Physiology
  • Transuterine microinjection
  • Ventral laparotomy
  • Video microscopy

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)

Cite this

Gene transfer to the developing mouse inner ear by in vivo electroporation. / Wang, Lingyan; Jiang, Han; Brigande, John.

In: Journal of Visualized Experiments, No. 64, e3653, 30.06.2012, p. 1.

Research output: Contribution to journalArticle

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