Fate-Mapping Technique: Targeted Whole-Embryo Electroporation of DNA Constructs into the Germ Layers of Mouse Embryos 7-7.5 Days Post-coitum.

ABSTRACT

INTRODUCTIONFate maps reveal body plan organization and presage the expression of molecular characteristics of cell lineages and formation of body parts. This protocol targets DNA expression constructs into the germ layers of gastrula-stage mouse embryos by focal electroporation. Plasmids utilizing a promoter that drives widespread, non-lineage-restricted expression of transgenes are introduced to cells in defined germ layer regions by whole-embryo electroporation. Germ-layer cells are exposed to the DNA by microinjecting the plasmids into the proamniotic cavity (ectoderm) or directly into the intercellular space of the mesenchyme (mesoderm), or by incubating the embryo in the DNA solution (endoderm). Electroporation is performed on whole embryos in vitro by electric current-mediated permeation of the cell membrane, which allows DNA adsorbed to cell surfaces to enter the cells. A point electrode is used to focus the electric field to the intended site of electroporation and a plate electrode is used to generate the current at an effective voltage low enough to minimize damage to the embryonic tissue. Expression of the transgene can be used to track the fate and movement of cells and the cDNA to study the functional consequences of overexpression of genes during embryonic development in vitro.