Chick Embryo Culture and Electroporation

Yukinori Endo1

1 Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 19.15
DOI:  10.1002/0471143030.cb1915s56
Online Posting Date:  September, 2012
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Important events in embryonic development such as gastrulation, neurulation, and cranial neural crest development occur in ectodermal tissues during vertebrate embryonic development. Although the chicken embryo is a well‐established model system in developmental biology, problems of accessibility of the ectoderm for experimental manipulation and an inability to generate gene knockouts previously impeded studies of gene regulation and key processes during chicken gastrulation and neurulation. The technique of in ovo electroporation permits genetic manipulation and provides a powerful animal model. However, the problem of accessibility to the ectoderm in ovo requires an ex ovo whole‐embryo culture approach combined with electroporation. This unit provides convenient and reproducible whole‐embryo ex ovo culture and electroporation protocols. These chicken embryo culture protocols can be used not only for gene regulatory experiments, but also for time‐lapse imaging of the dynamics of early vertebrate development. Curr. Protoc. Cell Biol. 56:19.15.1‐19.15.10. © 2012 by John Wiley & Sons, Inc.

Keywords: chicken embryo; ex ovo culture; electroporation; morpholino; neural crest cells

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Table of Contents

  • Introduction
  • Basic Protocol 1: Ex Ovo Whole‐Chicken Embryo Culture
  • Basic Protocol 2: Electroporation of Morpholino Oligonucleotides or Plasmids Into Chicken Embryos
  • Basic Protocol 3: Time‐Lapse Movies
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Ex Ovo Whole‐Chicken Embryo Culture

  • Chicken eggs (e.g., Charles River Laboratories)
  • 70% ethanol
  • Hanks' balanced salt solution (HBSS, see recipe)
  • 37° to 38°C humidified incubator
  • 10‐cm disposable plastic Petri dishes
  • L‐shaped bent spoon (∼1‐ to 3‐ml capacity, Fig. F)
  • 7.5‐ml disposable transfer pipets (VWR, cat. no. 414004‐005)
  • 50‐ml disposable polypropylene conical tubes
  • Kimwipes
  • Filter paper (Fig. A, autoclaved)
  • Forceps and fine scissors
  • Pasteur pipets (53/ 4‐in. or 14.5‐cm length, autoclaved)
  • Parafilm (5 × 5–cm pieces)
  • 35‐mm disposable plastic Petri dishes (Fig. B)
  • 150‐mm glass Petri dishes with lids
  • Razor blade
  • Dissecting microscope (e.g., Stemi SV6, Zeiss)

Basic Protocol 2: Electroporation of Morpholino Oligonucleotides or Plasmids Into Chicken Embryos

  • HBSS (see recipe)
  • HH4‐5 embryo mounted on filter paper (see protocol 1)
  • Standard control morpholino or custom morpholino oligonucleotides (GeneTools; see recipe)
  • DNA plasmids (see recipe)
  • Electroporator (Protech International, cat. no. CUY21)
  • Electrode chamber with a negative electrode (Protech International, cat. no. CUY700‐P20E; Fig. C)
  • Positive electrode (Protech International, cat. no. CUY701‐P2L; Fig. C)
  • Foot switch (Protech International, cat. no. C200)
  • Glass disposable Pasteur pipets (53/ 4‐in. or 14.5‐cm length, autoclaved)
  • Femtotip II microinjection capillaries (Eppendorf)
  • Forceps
  • Dissecting microscope (e.g., Stemi SV6, Zeiss)

Basic Protocol 3: Time‐Lapse Movies

  • Chicken embryos at HH4‐7 on filter paper (see protocol 1)
  • Thin albumen (see protocol 1)
  • MatTek glass‐bottom dish (MatTek Corporation, cat. no. P50G‐1.5‐14‐F)
  • Parafilm (5 × 5–cm pieces)
  • Inverted microscope (e.g., Axiovert 40C, Zeiss) equipped with a humidified CO 2 chamber, CCD camera, and time‐lapse recording software (e.g., Infinity 2 cool scan camera time‐lapse system, Lumenara)
  • 5× and/or 10× objective lens
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Literature Cited

Literature Cited
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   Chapman, S.C., Collignon, J., Schoenwolf, G.C., and Lumsden, A. 2001. Improved method for chick whole‐embryo culture using a filter paper carrier. Dev. Dyn. 220:284‐289.
   Endo, Y., Osumi, N., and Wakamatsu, Y. 2002. Bimodal functions of Notch‐mediated signaling are involved in neural crest formation during avian ectoderm development. Development 129:863‐873.
   Hamburger, V. and Hamilton, H.L. 1951. A series of normal stages in the development of the chick embryo. J. Morphol. 88:49‐92.
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   Muramatsu, T., Nakamura, A., and Park, H.M. 1998. In vivo electroporation: A powerful and convenient means of nonvial gene transfer to tissues of living animals. Int. J. Mol. Med. 1:55‐62.
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   Taneyhill, L.A., Coles, E.G., and Bronner‐Fraser, M. 2007. Snail2 directly represses cadherin6B during epithelial‐to‐mesenchymal transitions of the neural crest. Development 134:1481‐1490.
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