Stowers Institute for Medical Research, 1000 E. 50th Street, Kansas City, MO 64110, USA.
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.
Dev Biol. 2019 Apr 1;448(1):7-15. doi: 10.1016/j.ydbio.2019.01.005. Epub 2019 Jan 11.
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.
要深入理解进化发育生物学的机制，就需要开发新的技术，以便在系统发育上差异很大的生物体中操控基因功能。最近，通过注射短发夹 RNA（shRNA）实现基因的特异性敲低已应用于海葵 Nematostella vectensis，表明 shRNA 方法可用于高效、稳健的基因特异性敲低。然而，显微注射这种耗时耗力的方法限制了该技术的应用及其在大规模实验中的应用。为了解决这个问题，我们在此提出了一种将 shRNA 递送至海葵卵的电穿孔方案。该方法利用电穿孔的速度和简易性，使用户能够在数分钟内处理数百个卵或胚胎中的基因表达。我们详细描述了实验程序，包括试剂、电穿孔条件、海葵卵的制备和实验动物的后续护理。最后，我们演示了几种具有已知表型的内源性和外源性基因的敲低，并讨论了该方法的潜在应用。
