title: Nonviral gene transfer to surface skin of mid-gestational murine embryos by intraamniotic injection and subsequent electroporation.
abstract: The surface epithelium of mid-gestational murine embryos is thought to be an attractive target for gene therapy in vivo, due to its visibility and accessibility from the external surface of the maternal uterus. Almost all studies of in utero gene transfer have adopted viral vectors for infection of fetal epithelium, and depended on intraamniotic introduction and simple incubation of vectors, leading to only infection of the surface layer (periderm) of fetal skin. Here we report a simple and convenient method of gene transfer of plasmid DNA into the deeper portion of surface skin of murine mid-gestational fetus. One to two microlitres of a solution containing a lacZ expression plasmid (0.5-1 microg) and trypan blue (0.05%) were placed onto the surface of a fetus (E 14.5) near the eye by a micropipette attached to a mouthpiece. This fetus was immediately electroporated by placing it between tweezer-type electrodes attached to a square-pulse generator. At 1 and 4 days after gene transfer, fetuses were subjected to histochemical staining for lacZ activity in the presence of X-Gal, a substrate for lacZ. Focal reactions were observed in the skin epidermal layers including periderm and basal layer 1 day after DNA introduction. However, lacZ-positive cells were limited to a skin surface layer, the stratum corneum, in the samples obtained 4 days after gene transfer. Similar observation was also made in the transgenic fetuses (carrying a lacZ gene placed immediately downstream of the loxP-flanked sequence) injected with Cre expression vector. These findings suggest rapid movement of fetal epidermal cells toward the surface during late developmental stages. This local gene transfer approach appears to be effective as a method for skin-targeted gene transfer, enabling study of the role of genes of interest and tracing of cell lineage during fetal skin development.
authors.affiliation: The Institute of Medical Sciences, Tokai University, Bohseidai, Isehara, Kanagawa, Japan. masasato@is.icc.u-tokai.ac.jp
authors.forename: Masahiro
authors.initials: M
authors.lastname: Sato
authors.forename: Maya
authors.initials: M
authors.lastname: Tanigawa
authors.forename: Natsuko
authors.initials: N
authors.lastname: Kikuchi
mesh_terms: D000650:Amnion; D000818:Animals; D018274:Electroporation; D004622:Embryo, Mammalian / Q000378:metabolism*; D005333:Fetus / Q000378:metabolism; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D007267:Injections; D007763:Lac Operon; D051379:Mice; D012867:Skin / Q000378:metabolism*; D014162:Transfection