Holmes Katie E, Wyatt Matthew J, Shen Yu-chi, Thompson Deborah A, Barald Kate F
Department of Veterinary Science, University of Wisconsin, Madison, USA.
J Vis Exp. 2011 Jan 7(47):2466. doi: 10.3791/2466.
In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.
近年来，电穿孔已成为一种将DNA、RNA和吗啉代寡核苷酸体内转染到包括眼睛、大脑和斑马鱼体节在内的各种组织的常用技术。与其他基因操作方法相比，电穿孔的优势在于，通过施加电流，可以在空间和时间上对特定组织进行靶向，以导入大分子。在此，我们描述了利用电穿孔将mif和mif样吗啉代寡核苷酸转染到斑马鱼发育中的内耳组织中的方法。在过去的研究中，在1至8细胞期将mif吗啉代寡核苷酸注射到胚胎中会导致神经系统、眼睛以及耳朵出现广泛的形态学变化。通过在发育后期靶向内耳组织，我们可以确定MIF在发育中的内耳中的主要作用，而不是其他组织影响可能产生的次要作用。通过使用鬼笔环肽和乙酰化微管蛋白染色来研究与后黄斑相关的神经元、神经突和毛细胞的形态，我们能够评估电穿孔作为斑马鱼内耳靶向转染方法的有效性。将吗啉代寡核苷酸注射到24hpf胚胎的耳囊中并进行电穿孔，然后与未处理、仅注射或仅电穿孔的胚胎进行比较。与对照组相比，注射并电穿孔的胚胎毛细胞数量减少，听神经节（SAG）的神经支配减少，SAG神经元数量减少。我们的结果表明，在后期将吗啉代寡核苷酸直接递送至耳囊中可避免在1-8细胞期递送吗啉代寡核苷酸时产生的非特异性神经系统和神经嵴效应。它还允许检查直接针对内耳的效应，而不是大脑、神经嵴或耳周间充质的主要效应对内耳产生的次要效应。
