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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Abstract

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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Materials

Basic Protocol 1: Ex Ovo Whole‐Chicken Embryo Culture

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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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Figures

  •   FigureFigure 19.15.1 (A) Whatman 3MM CHR filter paper with scissors to a 2 × 2‐cm square, with the center removed using a 6.0‐mm diameter paper hole puncher. The paper provides support and tension for the vitelline membrane and the embryo. (B) A 35‐mm disposable plastic tissue culture dish with its lid facing upward at its bottom, covered firmly by two layers of Parafilm; a triangular (5‐ to 7‐mm) hole is cut in its center using a razor blade. (C) Thin and thick (viscous) albumen of the yolk. (D) An embryo mounted onto a filter paper is detached from the yolk using forceps pulled in an oblique direction (red arrow). (E) Embryos on 35‐mm dishes filled with thin albumen were then placed into a 150‐mm humidified glass Petri dish. (F) Photograph of L‐shape bent spoons.
  •   FigureFigure 19.15.2 (A) Schematic top view of electroporation chamber with a negative electrode. (B) Side view of electroporation chamber showing negative and positive electrodes. (C) Photograph of an electroporation chamber and electrode used for electroporation.
  •   FigureFigure 19.15.3 (A) HH17 chicken embryo on the filter membrane after 48‐hr culture from HH4. (B) Higher magnification view of the embryo in panel A. (C‐D) HH8 embryos transfected with FITC‐labeled control morpholino after 10.5 to 11 hr culture from HH4‐5.

Literature Cited

Literature Cited
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