In Ovo Electroporations of HH Stage 10 Chicken Embryos

Summary

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

Abstract

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology. With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function. By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function. Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements. Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive. This video demonstrates first how to make a window in the eggshell to manipulate the embryo. Next, the embryo is visualized with a dilute solution of India ink injected below the embryo. A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. Finally, the egg is sealed with tape and placed back into an incubator for further development. Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Protocol

Egg handling

1. Eggs may be kept at 13°C for up to one week prior to incubation without significant loss of viability (a small wine cooler is ideal for this purpose).
2. Prior to incubation, allow eggs to warm to room temperature, then place in a humidified chicken incubator set to 37.8°C (100°F).
3. We usually electroporate embryos approximately 48 hours from the start of incubation, when the embryo has reached Hamburger and Hamilton stage 10.
4. Time of incubation may vary according to desired Hamburger and Hamilton stage. Temperature is critical and deviation from optimal incubation temperature will alter incubation time and d....

Discussion

This protocol provides a step by step guide to neural tube electroporation of HH10 chick embryos. In addition to showing the technique, guidelines for storage and handling of eggs is also provided. This technique has a broad range of applications for genetic analysis and the ease and low cost of experiments makes them feasible for many labs.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chicken egg incubator			humidified, set to 37.80°C (100F).
Pulse generator	Genetronics	BTX T820	Square wave generator or comparable
Electrodes			our electrodes were made from 0.25 mm diameter platinum wire and were 5 mm long and spaced 1 mm apart
Connecter cables
Micromanipulator
Dissecting scope			with large working distance
Glass capillaries			and capillary puller or commercial glass needles
Leibovitz’s L-15 media
chicken eggs			fertilized
Curved scissors
10 ml syringe
Large bore needle			16-18G
Mouth pipette
India ink
Insulin syringe/syringe
Plasmid DNA			≥ 1μg/μl in sterile TE or 1X PBS containing 0.05% Fast Green dye