Isolation of highly pure and viable primordial germ cells from rainbow trout by GFP-dependent flow cytometry
Isolation of highly pure and viable primordial germ cells from rainbow trout by GFP-dependent flow cytometry
复制标题
DOI：
10.1002/mrd.20003
复制
发表时间：
2004-01-01
影响因子：
2.5
通讯作者：
Takeuchi, T
中科院分区：
文献类型：
--
作者：
Kobayashi, T;Yoshizaki, G;Takeuchi, T
一个高纯度和活的原始生殖细胞（PGC）人口似乎是一个重要的工具，建立一个细胞系，可以分化成生殖细胞谱系和研究鱼类PGC的分子生物学和生物化学。因此，本研究的目的是建立一种分离高纯度和活性PGCs的流式细胞术方法。作为PGC分离的材料，我们使用具有由鳟鱼vasa基因调控序列（pvasa-GFP）驱动的绿色荧光蛋白（GFP）基因的转基因虹鳟鱼。四个独立的转基因株进行荧光显微镜和GFP依赖的流式细胞仪分析。我们发现，除了PGC中的强荧光外，一些pvasa-GFP转基因株在体细胞中还显示出异位背景绿色荧光。虽然在四个pvasa-GFP转基因株生殖嵴体细胞的流式细胞仪分析显示了广泛的GFP强度，我们证明，体细胞污染的GFP阳性细胞群体显着减少，如果使用的转基因株没有异位背景绿色荧光。此外，前向光散射（FS）性质（其是相对细胞大小的指示）和侧向光散射（SS）性质（其由细胞形状和粒度确定）用于从GFP阳性细胞群体中去除非PGC污染物。通过分离具有高FS/SS值的GFP阳性细胞，我们能够有效地去除细胞泡和凋亡部分。因此，分离的PGC的纯度和存活率大大提高相比，使用GFP强度作为单一指标。因此，我们的流式细胞术方法，结合选择合适的转基因菌株没有异位背景绿色荧光，能够分离出高纯度和可行的PGC从虹鳟鱼。通过使用这种方法与细胞冷冻保存和细胞移植技术相结合，分离的PGCs还可用于保存濒危鱼类和具有商业价值性状的驯化鱼类的遗传资源。
A highly pure and viable primordial germ cell (PGC) population appears to be an essential tool for establishing a cell line that can differentiate into a germ cell lineage and for studying the molecular biology and biochemistry of fish PGCs. Therefore, the aim of the present study was to establish a flow cytometric method for isolating highly pure and viable PGCs. As the material for PGC isolation, we used transgenic rainbow trout possessing the green fluorescent protein (GFP) gene driven by trout vasa-gene regulatory sequences (pvasa-GFP). Four independent transgenic strains were subjected to fluorescence microscopy and GFP-dependent flow cytometric analyses. We found that some of the pvasa-GFP transgenic strains exhibited ectopic background green fluorescence in the somatic cells aside from strong fluorescence in PGCs. Although flow cytometric analysis of genital ridge somatic cells in the four pvasa-GFP transgenic strains revealed a wide range of GFP intensities, we proved that somatic cell contamination of the GFP-positive cell population was markedly reduced if transgenic strains without the ectopic background green fluorescence were used. In addition, the forward light-scattering (FS) property, which is an indication of relative cell size, and the side light-scattering (SS) property, which is determined by cell shape and granularity, were employed to remove non-PGC contaminants from the GFP-positive cell population. By isolating GFP-positive cells with high FS/SS values, we were able to effectively remove cell blebs and the apoptotic fraction. Consequently, the purities and survival rates of isolated PGCs were greatly improved compared with those using GFP intensity as a single indicator. Thus, our flow cytometric method, in combination with the selection of suitable transgenic strains without the ectopic background green fluorescence, is capable of isolating highly pure and viable PGCs from rainbow trout. By using this method in combination with cell-cryopreservation and cell transplantation techniques, the isolated PGCs may also be used for preserving the genetic resources of endangered fish species and domesticated fish strains carrying commercially valuable traits.