Genomic Integration and Germline Transmission of Plasmid Injected into Crustacean Daphnia magna Eggs
Genomic Integration and Germline Transmission of Plasmid Injected into Crustacean Daphnia magna Eggs
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DOI：
10.1371/journal.pone.0045318
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发表时间：
2012-09-18
期刊：
影响因子：
3.7
通讯作者：
Watanabe, Hajime
中科院分区：
文献类型：
--
作者：
Kato, Yasuhiko;Matsuura, Tomoaki;Watanabe, Hajime
水蚤，水蚤，几十年来一直是生态学，进化和环境科学的研究对象。在过去的几年中，已经确定了表达的序列标签和基因组序列。此外，已经建立了基于将RNA显微注射到卵中的过表达和基因沉默的功能方法。然而，这些方法的瞬时性质使我们无法分析发育后期的基因功能。为了克服这一限制，转基因将成为一个关键的工具。在这里，我们报告建立了一个转基因株系采用显微注射质粒到大型蚤卵。将绿色荧光蛋白（GFP）基因与D.在延伸因子1 α-1（EF 1 α-1）基因的启动子/增强子区控制下的大组蛋白H2 B基因，EF 1 α-1：：H2 B-GFP，被用作提供活性染色质的高分辨率可视化的报道基因。转基因系从注射后存活的总可育成虫的0.67%获得。其中一只转基因动物在胚胎发生和卵子发生期间在细胞核中显示荧光，在头对尾阵列中有两个EF 1 α-1：：H2 B-GFP拷贝。这是第一次报告的转基因技术在水蚤和新兴的基因组序列，将有助于推进知识的水蚤的分子生物学。
The water flea, Daphnia, has been the subject of study in ecology, evolution, and environmental sciences for decades. Over the last few years, expressed sequence tags and a genome sequence have been determined. In addition, functional approaches of overexpression and gene silencing based on microinjection of RNAs into eggs have been established. However, the transient nature of these approaches prevents us from analyzing gene functions in later stages of development. To overcome this limitation, transgenesis would become a key tool. Here we report establishment of a transgenic line using microinjection of plasmid into Daphnia magna eggs. The green fluorescent protein (GFP) gene fused with the D. magna histone H2B gene under the control of a promoter/enhancer region of the elongation factor 1 alpha-1 (EF1 alpha-1) gene, EF1 alpha-1::H2B-GFP, was used as a reporter providing high resolution visualization of active chromatin. Transgenic lines were obtained from 0.67% of the total fertile adults that survived the injections. One of the transgenic animals, which exhibited fluorescence in the nuclei of cells during embryogenesis and oogenesis, had two copies of EF1 alpha-1::H2B-GFP in a head-to-tail array. This is the first report of a transgenesis technique in Daphnia and, together with emerging genome sequences, will be useful for advancing knowledge of the molecular biology of Daphnia.