Isolation of teleost primordial germ cells using flow cytometry
Isolation of teleost primordial germ cells using flow cytometry
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DOI：
10.1387/ijdb.092914rg
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发表时间：
2010-01-01
影响因子：
0.7
通讯作者：
Yamaha, Etsuro
中科院分区：
文献类型：
--
作者：
Goto-Kazeto, Rie;Saito, Taiju;Yamaha, Etsuro
原始生殖细胞（PGCs）产生配子，配子是唯一能够将遗传信息传递给下一代的细胞。先前的报道表明，绿色荧光蛋白（gfp）与斑马鱼nos 13 ' utr mRNA的融合结构可用于标记许多鱼类的PGCs。在这里，我们试图利用这种标记策略，通过流式细胞术（FCM）分离硬骨鱼PGCs，并使用这些分离的PGCs来检测生殖细胞向性腺区域的迁移。在斑马鱼、medaka和金鱼中，通过将gfp-nos 1 3'UTR mRNA注射到1-4个细胞胚胎中来标记PGCs。当胚胎发育到体细胞发生或后期阶段时，将其酶解并使用流式细胞术分离出GFP阳性细胞。不同物种的PGCs聚集在胚胎细胞总数的FCM散点图的相同片段上，通过绘制前向散射强度与GFP强度的关系。原位杂交表明，分选后的斑马鱼细胞细胞质中表达vasa RNA，表明它们是PGCs。在体内移植实验中检测了斑马鱼细胞的迁移能力，大约30%的细胞迁移到宿主胚胎的性腺区域。这些观察结果表明，可以在不使用转基因鱼的情况下分离出PGCs，并且分离出的PGCs保留了迁移能力。我们的数据表明，该技术将对从一系列鱼类中分离PGCs具有价值。
Primordial germ cells (PGCs) generate gametes, the only cells that can transmit genetic information to the next generation. A previous report demonstrated that a fusion construct of green fluorescent protein (gfp) and zebrafish nos 1 3'UTR mRNA could be used to label PGCs in a number of fish species. Here, we sought to exploit this labeling strategy to isolate teleost PGCs by flow cytometry (FCM), and to use these isolated PGCs to examine germ cell migration to the gonadal region. In zebrafish, medaka and goldfish, the PGCs were labeled by injecting the gfp-nos 1 3'UTR mRNA into 1-4 cell embryos. When the embryos had developed to the somitogenesis or later stages, they were enzymatically disaggregated and GFP positive cells isolated using FCM. PGCs in the different species clustered in the same segments of the FCM scatter diagrams for total embryonic cells produced by plotting the forward scatter intensity against GFP intensity. In situ hybridization showed that the sorted zebrafish cells expressed vasa RNA in their cytoplasm, suggesting that they were PGCs. When the migration ability of the sorted cells from zebrafish was examined in an in vivo transplantation experiment, approximately 30% moved to the gonadal region of host embryos. These observations demonstrate that PGCs can be isolated without use of transgenic fishes and that the isolated PGCs retain the ability to migrate. Our data indicate that this technique will be of value for isolating PGCs from a range of fish species.