Introduction of cell markers into germ layer tissues of the mouse gastrula by whole embryo electroporation.
We have optimized the technique of electroporation for introducing genetic markers into cells of the gastrulating mouse embryo to follow cell fates, tissue movement, and lineage differentiation. Using a plate-needle electrode combination and specific route of plasmid delivery, labeling could be targeted to discrete regions of the epiblast or the endoderm of the late gastrula. Among the various types of fluorescent and chromogenic reporter constructs tested, those driven by CMV promoter are efficient and strong expression can be detected as soon as 2-3 h after electroporation. The efficacy of marking cell lineages by CRE-mediated activation of reporters proved to be inefficient for tracking cell lineages due to an obligatory 8-9-h lag from the electroporation of constructs to the expression of reporter. This significant time lag also raises concern of the temporal precision at which tissue- or stage-specific knock-out or activation of genetic activity may be achieved by the Cre-loxP mechanism.
Davidson
Bruce P
BP
Embryology Unit, Children's Medical Research Institute, Wentworthville, NSW, Australia.
Tsang
Tania E
TE
Khoo
Poh-Lynn
PL
Gad
Jacqueline M
JM
Tam
Patrick P L
PP
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015415:Biomarkers; D018929:Cell Culture Techniques; D019070:Cell Lineage; D018274:Electroporation; D004622:Embryo, Mammalian / Q000033:anatomy & histology; D005775:Gastrula / Q000502:physiology*; D017930:Genes, Reporter; D005821:Genetic Techniques; D005855:Germ Layers / Q000502:physiology*; D049452:Green Fluorescent Proteins; D019426:Integrases / Q000378:metabolism; D007763:Lac Operon / Q000502:physiology; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D014764:Viral Proteins / Q000378:metabolism
D015415:Biomarkers; D008164:Luminescent Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; C045073:Cre recombinase; D019426:Integrases; D000469:Alkaline Phosphatase