Yuan Shiaulou, Sun Zhaoxia
Department of Genetics, Yale University School of Medicine, CT, USA.
J Vis Exp. 2009 May 7(27):1113. doi: 10.3791/1113.
An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.
研究基因在发育过程中作用的一个重要工具是能够进行基因敲低、过表达和错误表达研究。在斑马鱼（Danio rerio）中，将RNA、DNA、蛋白质、反义寡核苷酸和其他小分子显微注射到发育中的胚胎中，为研究人员提供了一种在体内探索基因功能的快速且可靠的方法。在本视频文章中，我们将演示如何制备体外合成的EGFP mRNA以及针对与常染色体显性多囊肾病（ADPKD）相关的基因pkd2的翻译阻断吗啉代寡核苷酸，并将其显微注射到单细胞期斑马鱼胚胎中。然后，我们将通过验证GFP表达和表型分析来分析mRNA和吗啉代显微注射的成功率。该技术的广泛应用包括生成转基因动物和种系嵌合体、细胞命运图谱绘制和基因筛选。在此，我们描述了一种通过mRNA和吗啉代寡核苷酸注射来实现EGFP过表达和pkd2基因敲低的方案。
