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Chick embryo culture and electroporation.

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.

SUBMITTER: Endo Y

PROVIDER: S-EPMC3464100 | biostudies-other | 2012 Sep

REPOSITORIES: biostudies-other

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Chick embryo culture and electroporation.

Endo Yukinori Y

Current protocols in cell biology 20120901

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 ...[more]

PMID: 22968841

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Project description:BackgroundRetinal cell development has been extensively investigated; however, the current knowledge of dynamic morphological and molecular changes is not yet complete.ResultsThis study was aimed at revealing the dynamic morphological and molecular changes in retinal cell development during the embryonic stages using a new method of targeted retinal injection, in ovo electroporation, and immunohistochemistry techniques. A plasmid DNA that expresses the green fluorescent protein (GFP) as a marker was delivered into the sub-retinal space to transfect the chick retinal stem/progenitor cells at embryonic day 3 (E3) or E4 with the aid of pulses of electric current. The transfected retinal tissues were analyzed at various stages during chick development from near the start of neurogenesis at E4 to near the end of neurogenesis at E18. The expression of GFP allowed for clear visualization of cell morphologies and retinal laminar locations for the indication of retinal cell identity. Immunohistochemistry using cell type-specific markers (e.g., Visinin, Xap-1, Lim1+2, Pkcalpha, NeuN, Pax6, Brn3a, Vimentin, etc.) allowed further confirmation of retinal cell types. The composition of retinal cell types was then determined over time by counting the number of GFP-expressing cells observed with morphological characteristics specific to the various retinal cell types.ConclusionThe new method of retinal injection and electroporation at E3 - E4 allows the visualization of all retinal cell types, including the late-born neurons, e.g., bipolar cells at a level of single cells, which has been difficult with a conventional method with injection and electroporation at E1.5. Based on data collected from analyses of cell morphology, laminar locations in the retina, immunohistochemistry, and cell counts of GFP-expressing cells, the time-line and dynamic morphological and molecular changes of retinal cell development were determined. These data provide more complete information on retinal cell development, and they can serve as a reference for the investigations in normal retinal development and diseases.

Dataset Information

Chick embryo culture and electroporation.

Publications

Chick embryo culture and electroporation.

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