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View the entire protocol, "Fate-Mapping Technique: Targeted Whole-Embryo Electroporation of DNA Constructs into the Germ Layers of Mouse Embryos 7–7.5 Days Post-coitum," at BioSupplynet.com.
| P.-L. Khoo, V.J. Franklin, and P.P.L. Tam1 |
| Embryology Unit, Children's Medical Research Institute, University of Sydney, Wentworthville, NSW 2145, Australia |
| 1Corresponding author (ptam@cmri.usyd.edu.au) |
| Originally published in CSH Protocols; 2007; doi:10.1101/pdb.prot4893 |
ABSTRACT
Fate 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.
Products Available for this Protocol
| Protocol Material Description |
Product # |
Product Name |
Add to Cart |
| DMEM, with high glucose content |
D5796 |
Dulbecco’s Modified Eagle’s Medium - high glucose, With 4500 mg/L glucose, L-glutamine, and sodium bicarbonate, without sodium pyruvate, liquid, sterile-filtered, cell culture tested |
|
| Glutamine |
G6392 |
L-Glutamine, γ-irradiated, cell culture tested |
|
| Penicillin/streptomycin solution |
P7539 |
Penicillin–Streptomycin solution Hybri-Max™, liquid, sterile-filtered, hybridoma tested |
|
| Glucose |
G5146 |
D-(+)-Glucose, Hybri-Max™, powder, hybridoma tested |
|
| Sodium pyruvate for PB1 |
P5280 |
Sodium pyruvate, cell culture tested, insect cell culture tested, ≥99%, powder |
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| KCl |
P9541 |
Potassium chloride, for molecular biology, ≥99% |
|
| Phenol red-bicarbonate solution for PB1 |
319244 |
Phenol Red sodium salt solution, 0.04 wt. % in H2O |
|
| NaCl |
S3014 |
Sodium chloride, for molecular biology, ≥98% (titration) |
|
| Na2HPO4 • 12H2O |
71649 |
Sodium phosphate dibasic dodecahydrate, BioUltra, ≥99.0% |
|
| KH2PO4 |
P9791 |
Potassium phosphate monobasic, for molecular biology, ≥98% |
|
| CaCl2 • 2H2O |
C8106 |
Calcium chloride, meets USP testing specifications |
|
| MgCl2 • 6H2O |
M2393 |
Magnesium chloride hexahydrate, cell culture tested, insect cell culture tested |
|
| RS (rat serum) |
R9759 |
Rat serum |
|
| Paraffin oil, light |
34920 |
Paraffin oil, SPECTRANAL®, liquid |
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| DNA expression plasmid |
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| NaOH |
S8045 |
Sodium hydroxide, SigmaUltra, ≥98%, pellets (anhydrous) |
|
| Paraformaldehyde |
P6148 |
Paraformaldehyde, reagent grade, crystalline |
|
| Bovine serum albumin |
A4161 |
Albumin from bovine serum, cell culture tested, powder |
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