Gene transfer into cultured mammalian embryos by electroporation

To gain a better understanding of mammalian development at the molecular le vel, technology is needed that allows the transfer of exogenous genes into desired embryonic regions at defined stages of development. Our strategy ha s been to use electroporation (EP) of plasmid DNA following whole-embryo cu lture (WEC). In our gene transfer system, postimplantation rodent embryos a re taken out of the uterus and a purified DNA solution of mammalian express ion plasmid constructs is injected into the neural tube. A square-pulse cur rent is delivered using an electroporator with an optimizer. Electroporated embryos are allowed to develop in the WEC system for 24-48 h. Within the t argeted area, the proportion of transfected cells varied from 10% to approx imately 100% depending on the test conditions (e.g., DNA concentration, vol tage, duration of EP, and pulse number). The EP-WEC system has several adva ntages including rapid gene expression, minimal laboratory work, precisely targeted regions, and no risk for human beings. Application of the method i s useful in improving our understanding of early neural development (E7-E12 in mice), e.g., alteration of gene function via ectopic expression, interf erence with dominant negative proteins, and fate mapping with marker genes. In addition, EP can complement genetic approaches such as the generation o f knockout and transgenic mice. (C) 2001 Academic Press.