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Development of Tools for RNA interference (RNAi) of the engrailed Gene in Daphnia magna
Author(s) -
Singer Davi,
Beckman Matt
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.722.13
Subject(s) - engrailed , biology , rna interference , daphnia pulex , daphnia magna , daphnia , genetics , gene knockdown , gene , gene expression , rna , zoology , homeobox , chemistry , organic chemistry , toxicity , crustacean
The legend of the cyclops has been a part of international folklore for centuries. Yet, we do not fully understand the developmental genetic basis for cyclopia. Daphnia magna , a water flea, is a naturally occurring cyclops which can serve as a model to study this phenomenon. In this study tools were created to examine the role of the engrailed gene in Daphnia eye development. In Drosophila melanogaster (fruit flies), silencing of the engrailed gene affects segmentation during early development. Additional studies suggest that engrailed is one candidate gene involved in eye field development. To understand the role of engrailed in Daphnia development, we sought to knockdown the gene's expression using RNA interference (RNAi). Using the online databases Uniprot and the wFleaBase we acquired the D. magna hedgehog mRNA sequence. Using this sequence we designed primers to amplify portions of the D. magna engrailed gene. Engrailed gene PCR products were then cloned into the pGC‐Blue vector. Work continues to clone the engrailed insert into the RNAi feeding vector L4440. The L4440 plasmid will be transformed into HT115 E.coli and fed to Daphnia . Future work will also include qPCR studies of the engrailed gene expression during key time‐points in Daphnia eye development and following RNAi knockdown. In summary, through this work tools are being developed to study the role of engrailed in cyclopean eye development. We believe that by leveraging in our studies, Daphnia magna , a true cyclopean organism that evolution built, we can gain a deeper understanding of the developmental genetic basis for midline disorders. Support or Funding Information We thank the Augsburg Biology Department, Augsburg McNair Program and the Augsburg LSAMP STEM program for supporting this work.