Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

Summary

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

Abstract

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos¹. Different structures such as DNA plasmids encoding genes^2-4, small interfering RNA (siRNA) plasmids⁵, small synthetic RNA oligos⁶, and morpholino antisense oligonucleotides⁷ can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)⁸ and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.

In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes⁹ and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos^10-12.

Protocol

1. Ex ovo Culture

1. Fresh fertilized eggs are laid onto their long side in a forced-draft incubator (BSS160, Ehret, Germany) at 37.5 °C with 60% humidity for incubation. Eggs stored at 12 °C for longer than one week should not be used.
2. After incubation for 2.5 days (about stage HH17), take out the eggs and label the top with a pencil to indicate direction for cracking.
3. Before cracking, pour about 20 ml sterile distilled water into a clean large Petri dish (diameter of 145 mm; Greiner Bio-One GmbH, Germany).
4. Place another small sterile Petri dish (diameter of 94 mm; Greiner Bio-One GmbH) into the large Petr....

Discussion

This protocol provides a guide for gene transfer into older chicken embryos (e.g, into the optic tectum at E6) by ex ovo electroporation. This method extends the use of electroporation to older chicken embryos and can be easily applied to many laboratories for studying gene function in vivo at later stages. Embryos from E4 to at least E7 can be successfully electroporated by this method and the electroporated embryos can be survival until E15¹¹. Besides the brain, this method can be also used.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Electroporator	Nepa Gene, Japan	CUY21-Edit
Electrodes	Nepa Gene, Japan	CUY661-3x7
Egg incubator	Ehret, Germany	BSS160
Embryo incubator	BINDER GmbH, Germany
Fluorescent microscope	Keyence, Deutschland GmbH	BZ-8000
Petri dish	Greiner Bio-One GmbH, Germany
Micr–lectrode puller	World Precision Instruments, Germany	PUL-100