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.

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

“…Consistent with a previous study 11 , GFP + cells were detected in embryos 1-2 hr after electroporation ( Figure 2E and 2G). When distal epiblast cells at the late primitive streak stage embryo were electroporated, labelled cells contributed to the neural ectoderm after 24 hr in culture ( Figure 2E and 2F).…”

“…For electroporation of nucleic acids in embryonic tissues, gold plated electrodes have most commonly been used, allowing targeting of cells in a broad spatial range [7][8][9] . To achieve a more localized gene transfer, a needle-shaped electrode has been utilized to achieve a focal electric field 10,11 . Using this method, the authors showed that after the electroporation, around 30-60 cells had taken up the DNA construct 11 .…”

“…To achieve a more localized gene transfer, a needle-shaped electrode has been utilized to achieve a focal electric field 10,11 . Using this method, the authors showed that after the electroporation, around 30-60 cells had taken up the DNA construct 11 . Nevertheless, it seems that accurately adjusting the number of electroporated cells remains difficult with a fixed-width electrode.…”

A microdevice to locally electroporate embryos with high efficiency and reduced cell damage

“…Fluorescent protein expression was detected in one to four VE cells 4 h after plasmid electroporation (Fig. 4D, n=14), a timing in agreement with previous observations (Davidson et al, 2003;Khoo et al, 2007). Prominent apical vacuoles and basal projections in migrating DVE cells were observed when cytoplasmic or membranetargeted fluorescent proteins were used ( Fig.…”

“…This limitation has been overcome by replacing one of the plates by a needle (Fig. 1C), thereby creating a significant variation of the electric potential over a small area (Davidson et al, 2003;Khoo et al, 2007). Importantly, permeation is efficiently restricted in space only if the needle is close enough to the targeted tissue, which may result in cell damage.…”