用于斑马鱼转基因的修饰细菌人工染色体

Modified bacterial artificial chromosomes for zebrafish transgenesis.

作者信息

Yang Zhongan, Jiang Hong, Chaichanasakul Thawinee, Gong Shiaoching, Yang Xiangdong William, Heintz Nathaniel, Lin Shuo

机构信息

Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, CA 90095-1606, USA.

出版信息

Methods. 2006 Jul;39(3):183-8. doi: 10.1016/j.ymeth.2006.04.011.

DOI:10.1016/j.ymeth.2006.04.011

PMID:16828309

Abstract

Transgenesis using bacterial artificial chromosomes (BAC) offers greater fidelity in directing desirable expression of foreign genes. Application of this technology in the optically transparent zebrafish with fluorescent protein reporters enables unparalleled visual analysis of regulation of gene expression in a living organism. Here we describe a streamlined procedure of direct selecting multiple BAC clones based on public sequence databases followed by rapid modification with GFP or RFP for transgenic analysis in zebrafish. Experimental procedures for BAC DNA preparation, microinjection of zebrafish embryos and screening of transgenic zebrafish carrying GFP/RFP modified BAC clones are detailed.

摘要

使用细菌人工染色体（BAC）进行转基因能够更忠实地指导外源基因的理想表达。将该技术应用于带有荧光蛋白报告基因的光学透明斑马鱼，可对活生物体中的基因表达调控进行无与伦比的可视化分析。在此，我们描述了一种简化程序，即基于公共序列数据库直接筛选多个BAC克隆，随后用绿色荧光蛋白（GFP）或红色荧光蛋白（RFP）进行快速修饰，用于斑马鱼的转基因分析。文中详细介绍了BAC DNA制备、斑马鱼胚胎显微注射以及筛选携带GFP/RFP修饰BAC克隆的转基因斑马鱼的实验步骤。