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Dev Biol. 2019 Jan 11. pii: S0012-1606(18)30633-X. doi: 10.1016/j.ydbio.2019.01.005. [Epub ahead of print]

Electroporation of short hairpin RNAs for rapid and efficient gene knockdown in the starlet sea anemone, Nematostella vectensis.

Author information

1
Stowers Institute for Medical Research, 1000 E. 50th Street, Kansas City, MO 64110, USA.
2
Stowers Institute for Medical Research, 1000 E. 50th Street, Kansas City, MO 64110, USA; Dept. Anatomy and Cell Biology, University of Kansas School of Medicine, Kansas City, KS 66160 USA. Electronic address: mg2@stowers.org.

Abstract

A mechanistic understanding of evolutionary developmental biology requires the development of novel techniques for the manipulation of gene function in phylogenetically diverse organismal systems. Recently, gene-specific knockdown by microinjection of short hairpin RNA (shRNA) was applied in the sea anemone Nematostella vectensis, demonstrating that the shRNA approach can be used for efficient and robust sequence-specific knockdown of a gene of interest. However, the time- and labor-intensive process of microinjection limits access to this technique and its application in large scale experiments. To address this issue, here we present an electroporation protocol for shRNA delivery into Nematostella eggs. This method leverages the speed and simplicity of electroporation, enabling users to manipulate gene expression in hundreds of eggs or embryos within minutes. We provide a detailed description of the experimental procedure, including reagents, electroporation conditions, preparation of Nematostella eggs, and follow-up care of experimental animals. Finally, we demonstrate the knockdown of several endogenous and exogenous genes with known phenotypes and discuss the potential applications of this method.

PMID:
30641041
DOI:
10.1016/j.ydbio.2019.01.005
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