Summary
Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.
Abstract
A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.
Protocol
Discussion
The method described above is a modification of a previously described technique for delivering lipophilic dyes to small cell subsets or single progenitors 3. It has three main advantages over the standard electroporation technique. First, it provides a means for labeling with confidence discrete sites in target tissues, allowing much greater spatial resolution than electroporation (see for example Figure 1B,C). Second, it permits labeling of single cells, thus allowing to trace their lineage in-vivo …
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Shlomi Krispin for the neural tube image. In particular, we acknowledge Yuval Cinnamon for initiating the GFP transfection technique. This work was supported by grants from the Israel Science Foundation, Association Francaise contre les Myopathies, and Deutche Forschungsgemeinshaft, Collaborative Research Centre 488 to C.K.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Pipette tubes | Sutter Instrument | B100-75-10 | ||
| Capillaries | Drummond Scientific | 2-000-100 | ||
| Insect needles | Watkins & Doncaster | E6871 | ||
| Pen-Strep solution | Biological Industries | 03-031-1B |
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