Visualization of Primordial Germ Cells in Transgenic Rainbow Trout Carrying Green Fluorescent Protein Gene Driven by vasa Promoter
Visualization of Primordial Germ Cells in Transgenic Rainbow Trout Carrying Green Fluorescent Protein Gene Driven by vasa Promoter
复制标题
携带由 vasa 启动子驱动的绿色荧光蛋白基因的转基因虹鳟鱼原始生殖细胞的可视化
DOI：
10.2331/fishsci.68.sup2_1067
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发表时间：
2002
影响因子：
1.9
通讯作者：
Toshio Takeuchi
中科院分区：
文献类型：
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作者：
G. Yoshizaki;Y. Takeuchi;Haruo Tominaga;Terumasa Kobayashi;Toshio Takeuchi
为了在鱼类中开发细胞介导的基因转移技术，需要能够分化成生殖细胞谱系的细胞系。在这项研究中，我们开发了技术，以识别，可视化，并分离活的原始生殖细胞（PGCs）在体外培养，后来的基因转移工作。在许多动物中，vasa基因产物已被发现在生殖细胞谱系中，我们以前已经确定和其同源物（RtVLG）在虹鳟鱼的特点。鳟鱼胚胎在不同发育阶段的整装原位杂交显示，转录本定位于PGCs，这表明RtVLG转录可用作PGCs的标记。为了在体内鉴定PGC，我们构建了含有由RtVLG调控区驱动的绿色荧光蛋白（GFP）基因（pvasa-GFP）的质粒，将其显微注射到受精卵中，并饲养成熟的鱼。F1代转基因鳟鱼在各个发育阶段仅在PGCs中显示绿色荧光。该表达模式与内源性RtVLG基因的表达模式一致。为了获得用于体外培养的PGCs，从转基因胚胎中切下生殖嵴，用胰蛋白酶解离，并使用流式细胞仪将它们分选成GFP阳性和GFP阴性部分。RT-PCR分析显示RtVLG仅在GFP阳性细胞中表达。这些结果证实分离的GFP阳性细胞是PGC。我们目前正试图建立细胞系，以供进一步研究。
To develop a cell-mediated gene transfer technique in fish, a cell line that can differentiate into the germ cell lineage is needed. In this study, we developed techniques to identify, visualize, and isolate live primordial germ cells (PGCs) for in vitro culture and later for gene transfer work. In many animals, the vasa gene products have been found in the germ cell lineage, and we have previously identified and characterized its homologue (RtVLG) in rainbow trout. Whole mount in situ hybridization of trout embryos at various developmental stages revealed that transcripts were localized to PGCs, suggesting that RtVLG transcription can be used as a marker for PGCs. To identify PGCs in vivo, we constructed a plasmid containing the green fluorescent protein (GFP) gene driven by the RtVLG regulatory regions (pvasa-GFP), microinjected it into fertilized eggs, and raised mature fish. The F1 generation of these transgenic trout showed green fluorescence only in PGCs in all developmental stages. This expression pattern was consistent with that of the endogenous RtVLG gene. To obtain PGCs for in vitro culture, the genital ridges were excised from transgenic embryos, dissociated by trypsin, and a flow cytometer was used to sort them into GFP-positive and GFP-negative fractions. RT-PCR analysis revealed that RtVLG was expressed only in GFP-positive cells. These results confirm that the isolated GFP-positive cells are PGCs. We are presently attempting to establish cell lines for further work.