Introduction and expression of foreign genes in cultured mouse embryonic gonads by electroporation.
To analyse the functions of genes that are expressed and potentially involved in the development of embryonic gonad cells, a method was developed by which foreign genes can be introduced and expressed in cultured mouse genital ridges. Genital ridges from mouse embryos at 12.5 days post coitus (dpc) were injected with plasmid DNA of a green fluorescence protein (GFP) gene construct and then placed between small electrodes. Rectangular pulses were charged to electroporate DNA into the cells. The treated genital ridges were cultured on a membrane of a culture insert, and GFP gene expression was observed under a fluorescence microscope. Green fluorescence protein expression in the genital ridges was found as early as 1 h after electroporation. Thereafter, the expression gradually increased, peaked after 1 day, and then decreased. A significant number of cells were, however, still positive for fluorescence even after 2 weeks in the culture, in which both gonads and germ cells had continued to develop. The GFP gene was expressed in 1-2% of cells in each genital ridge in a DNA concentration-dependent manner. In addition, we confirmed that an electroporated red fluorescent protein (DsRed) gene construct was expressed in GFP-expressing primordial germ cells in genital ridges of Oct-4/GFP transgenic embryos, although the DNA was mainly found in somatic cells in genital ridges. Finally, an expression vector containing the internal ribosome entry site-green fluorescent protein (IRES-GFP) gene was constructed. An inserted lacZ gene showed similar expression pattern to that of GFP Using this vector, we can easily monitor the expression of an inserted gene of interest by GFP expression. Therefore, this experimental system could be useful for quick assays of gene function in genital ridges.
Nakamura
Yuri
Y
Department of Molecular Embryology, Research Institute, Osaka Medical Center for Maternal and Child Health, Izumi, Japan.
Yamamoto
Miwako
M
Matsui
Yasuhisa
Y
D000818:Animals; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics; D018274:Electroporation; D005260:Female; D015870:Gene Expression; D006066:Gonads / Q000196:embryology*; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D050814:Octamer Transcription Factor-3; D014157:Transcription Factors; D014162:Transfection; D000097573:Red Fluorescent Protein
D004268:DNA-Binding Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D050814:Octamer Transcription Factor-3; D014157:Transcription Factors; D000097573:Red Fluorescent Protein; C494174:Pou5f1 protein, mouse