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.
Egg handling
Preparation
Windowing
Optional: To visualize the neural tube, inject ink solution with 26 gauge needle underneath the embryo (insert needle under the embryo from outside the blood ring). India ink should be diluted 1:5 with Hanks or comparable sterile buffered solution.
Injection and Electroporation
Note: Be careful not to have tip larger then neural tube diameter. In general, you will be able to tell if the optimal tip size has been achieved by how easy or hard it is to pull liquid into the capillary. If there is extreme resistance, the opening is probably too small and the tip should be broken again higher up. If there is little or no resistance, the opening is too large and a new needle should be used.
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.
The authors have nothing to disclose.
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
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 |