Ando Hideki, Okamoto Hitoshi
Laboratory for Developmental Gene Regulation, Brain Science Institute, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama, 351-0198, Japan.
Mar Biotechnol (NY). 2006 May-Jun;8(3):295-303. doi: 10.1007/s10126-005-5138-6. Epub 2006 Apr 18.
Functional analyses of gene function by knockdown and expression approaches strongly enhance the genetic study of development. In vivo application of the introduction of inhibitors of gene expression, mRNA, and expression constructs in the target region make it possible to perform region- and stage-specific regulation of gene function in a simple manner. As a basic tool for the conditional regulation of gene expression in target tissue, we present methods for the efficient introduction of antisense morpholino oligonucleotide (MO), mRNA, and expression plasmid constructs into early and later stage zebrafish embryo and larva. Lipofection of a neuron-specific expression construct plasmid encoding green fluorescent protein (GFP) into optic vesicle resulted in clear GFP expression in the retinotectal pathway in hatched larva. Co-lipofection of MO and GFP mRNA to the presumptive head region resulted in brain-specific knockdown of the gene in mid-stage embryos.
通过基因敲低和表达方法对基因功能进行功能分析，极大地增强了发育遗传学研究。在目标区域体内应用基因表达抑制剂、mRNA和表达构建体，使得以简单的方式对基因功能进行区域和阶段特异性调控成为可能。作为在目标组织中进行基因表达条件性调控的基本工具，我们展示了将反义吗啉代寡核苷酸（MO）、mRNA和表达质粒构建体有效导入斑马鱼胚胎和幼体早期及后期阶段的方法。将编码绿色荧光蛋白（GFP）的神经元特异性表达构建体质粒通过脂质转染导入视泡，在孵化后的幼体视网膜 - 脑顶盖通路中产生了清晰的GFP表达。将MO和GFP mRNA共同脂质转染到假定的头部区域，导致中期胚胎中该基因在脑部特异性敲低。
