Establishment of medaka (Oryzias latipes) transgenic lines with the expression of green fluorescent protein fluorescence exclusively in germ cells:: A useful model to monitor germ cells in a live vertebrate
Establishment of medaka (Oryzias latipes) transgenic lines with the expression of green fluorescent protein fluorescence exclusively in germ cells:: A useful model to monitor germ cells in a live vertebrate
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DOI：
10.1073/pnas.041315498
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发表时间：
2001-02-27
影响因子：
11.1
通讯作者：
Nagahama, Y
中科院分区：
文献类型：
--
作者：
Tanaka, M;Kinoshita, M;Nagahama, Y
我们已经培育出转基因青鳉（硬骨鱼、Oryzias latipes），这使我们能够通过活体标本中的绿色荧光蛋白 (GFP) 荧光来监测生殖细胞。使用两种青鳉品系：himedaka（橙红色品种）和近交系 QurtE。通过显微注射含有青鳉vasa基因(olvas)推定启动子区域和3'区域的构建体来获得转基因系。在第 25 阶段（血液循环开始）左右，位于后肠腹外侧区域的原始生殖细胞 (PGC) 中 GFP 荧光强度显着增加。整体原位杂交和通过 GFP 荧光监测异位细胞表明 (i) PGC 规范后合子 olvas 表达增加，(ii) PGC 在 20-25 阶段后可以异位维持其细胞特征。在孵化当天左右，QurtE 菌株在 GFP 荧光生殖细胞数量上明显表现出性别二态性，这一发现与白细胞（QurtE 的性别特异性标记）的出现一致。 GFP 表达在成熟卵巢和睾丸的整个后期阶段持续存在。因此，这些转基因青鳉代表了一个活体脊椎动物模型，用于研究生殖细胞如何迁移以形成性二态性性腺，以及可能影响性腺发育的环境物质的潜在检测系统。还讨论了使用转基因构建体作为选择标记来在胚胎发生过程中有效分离种系传递创始人。
We have generated transgenic medaka (teleost, Oryzias latipes), which allow us to monitor germ cells by green fluorescent protein (GFP) fluorescence in live specimens. Two medaka strains, himedaka (orange-red variety) and inbred QurtE, were used. The transgenic lines were achieved by microinjection of a construct containing the putative promoter region and 3' region of the medaka vasa gene (olvas). The intensity of GFP fluorescence increases dramatically in primordial germ cells (PGCs) located in the ventrolateral region of the posterior intestine around stage 25 (the onset of blood circulation). Whole-mount in situ hybridization and monitoring of ectopically located cells by GFP fluorescence suggested that (i) the increase in zygotic olvas expression occurs after PGC specification and (ii) PGCs can maintain their cell characteristics ectopically after stages 20-25. Around the day of hatching, the QurtE strain clearly exhibits sexual dimorphisms in the number of GFP fluorescent germ cells, a finding consistent with the appearance of leucophores, a sex-specific marker of QurtE. The GFP expression persists throughout the later stages in the mature ovary and testis. Thus, these transgenic medaka represent a live vertebrate model to investigate how germ cells migrate to form sexually dimorphic gonads, as well as a potential assay system for environmental substances that may affect gonad development. The use of a transgenic construct as a selective marker to efficiently isolate germ-line-transmitting founders during embryogenesis is also discussed.