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Methods. 2001 May;24(1):35-42.

Gene transfer into cultured mammalian embryos by electroporation.

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1
Division of Biochemistry and Cell Biology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1, Ogawahigashi, Kodaira, Tokyo 187-8502, Japan. osumi@mail.cc.tohoku.ac.jp

Abstract

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

PMID:
11327800
DOI:
10.1006/meth.2001.1154
[Indexed for MEDLINE]
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