Nakamura Yuri, Yamamoto Miwako, Matsui Yasuhisa
Department of Molecular Embryology, Research Institute, Osaka Medical Center for Maternal and Child Health, Izumi, Japan.
Reprod Fertil Dev. 2002;14(5-6):259-65. doi: 10.1071/rd01130.
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.
为了分析在胚胎性腺细胞发育过程中表达且可能参与其中的基因的功能，开发了一种方法，通过该方法可将外源基因导入培养的小鼠生殖嵴并使其表达。将来自交配后12.5天（dpc）小鼠胚胎的生殖嵴注射绿色荧光蛋白（GFP）基因构建体的质粒DNA，然后置于小电极之间。施加矩形脉冲以将DNA电穿孔到细胞中。处理后的生殖嵴在培养插入物的膜上培养，并在荧光显微镜下观察GFP基因表达。电穿孔后1小时即可在生殖嵴中发现绿色荧光蛋白表达。此后，表达逐渐增加，在1天后达到峰值，然后下降。然而，即使在培养2周后，仍有大量细胞荧光呈阳性，在此期间性腺和生殖细胞都持续发育。GFP基因在每个生殖嵴中1-2%的细胞中以DNA浓度依赖性方式表达。此外，我们证实电穿孔的红色荧光蛋白（DsRed）基因构建体在Oct-4/GFP转基因胚胎生殖嵴中表达GFP的原始生殖细胞中表达，尽管DNA主要存在于生殖嵴的体细胞中。最后，构建了一个包含内部核糖体进入位点-绿色荧光蛋白（IRES-GFP）基因的表达载体。插入的lacZ基因显示出与GFP相似的表达模式。使用该载体，我们可以通过GFP表达轻松监测感兴趣的插入基因的表达。因此，该实验系统可用于快速检测生殖嵴中的基因功能。
