1Albrecht-Kossel-Institute for Neuroregeneration, School of Medicine University of Rostock, 2Institute of Anatomy I, School of Medicine University of Jena
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Luo, J., Yan, X., Lin, J., Rolfs, A. Gene Transfer into Older Chicken Embryos by ex ovo Electroporation. J. Vis. Exp. (65), e4078, doi:10.3791/4078 (2012).
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 genes2-4, 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 embryos10-12.
1. Ex ovo Culture
2. Preparation for ex ovo Electroporation
3. Gene Transfer into Chicken Optic Tectum by ex ovo Electroporation
4. Representative Results
Successful overexpression of the exogenous proteins of Cad7 and GFP by ex ovo electroporation is shown in Figure 1 as an example. When Cad7 plasmid together with GFP plasmid was electroporated into the tectum at E6, two or three days after electroporation the embryos were collected and fixed. At E8, the GFP protein is strongly expressed in the tectum in whole mount images (Figure 1A-C). Furthermore, in the sections of the tectum at E9, GFP protein (green in Figure 1D) and Cad7 protein (red in Figure 1E) are coexpressed (yellow in Figure 1F), suggesting that ex ovo electroporation is a successful method for gene transfer into the older chicken embryos in vivo.
Figure 1. When the plasmids encoding GFP and Cad7 are cotransfected into the chicken tectum at E6, two or three days after ex ovo electroporation, GFP protein (green) is strongly expressed as shown in wholemount images (A-C) at E8. In sections of the tectum at E9, GFP protein (green in D) and Cad7 protein (red in E) are coexpressed (yellow in F). BF, bright field image. Scale bar: 200 μm in A for A-C; 100 μm in D for D-F.
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 for gene transfer into the chicken limb system11.
Special attention should be paid to by ex ovo electroporation. For example, in order to obtain high survival of the embryos by ex ovo culture, eggs used should be fresh and stored less than one week at 12 °C; the small Petri dishes should be sterile and egg cracking performed at around E2.5. For ex ovo electroporation, the size and placement of electrodes should be correctly chosen according to the stage and targeted part of the embryos. When two plasmids are cotransfected, the injected plasmids should be in an appropriate ratio of the concentration (e.g., 1:8 between the marker GFP gene and targeted gene) in order to be sure that the GFP marked cells are almost coexpress the targeted gene.
No conflicts of interest declared.
This work was supported by a grant from the German Research Foundation (DFG; LU1455/1-1).
|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|
|Microelectrode puller||World Precision Instruments, Germany||PUL-100|