Single Cell Transfection in Chick Embryos

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

1. Preparation of Micropipettes

We pull our pipettes on a standard Narishige pp-83 pipette puller with a heat setting of 13.5. The precise setting must be calibrated, aiming at a pipette tip diameter of between 8 and 10 microns. We use filament-containing borosilicate glass tubes with an outer diameter of 1.0 mm and inner diameter of 0.75-0.78 mm. The pipettes are consequently stored in a plastic dish with the tips protected from contact with the edges of the dish.

2. DNA Preparation

The plasmid of interest is transfected into DH5α E. coli using standard procedures. Overnigh....

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Pipette tubes	Sutter Instrument Co.	B100-75-10
Capillaries	Drummond Scientific	2-000-100
Insect needles	Watkins Doncaster	E6871
Pen-Strep solution	Biological Industries	03-031-1B