Genomic integration and germline transmission of plasmid injected into crustacean Daphnia magna eggs

PLoS One. 2012;7(9):e45318. doi: 10.1371/journal.pone.0045318. Epub 2012 Sep 18.


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 1α-1 (EF1α-1) gene, EF1α-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 EF1α-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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions / genetics
  • Animals
  • Animals, Genetically Modified / genetics*
  • Blotting, Southern
  • Daphnia / genetics*
  • Green Fluorescent Proteins / genetics
  • Plasmids / genetics*
  • Promoter Regions, Genetic / genetics


  • 3' Untranslated Regions
  • Green Fluorescent Proteins

Grant support

This study was partly supported through a grant from the Ministry of Education, Culture, Sports, Science, and Technology (H.W.) and The Asahi Glass Fundation Rsearch Grant (H.W.). Y.K. is a Research Fellow of the Japan Society for the Promotion of Science (JSPS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.