Laplante Mary, Kikuta Hiroshi, König Melanie, Becker Thomas S
Sars Centre for Marine Molecular Biology, University of Bergen, High Technology Centre, Thormoehlensgate 55, 5008 Bergen, Norway.
Methods. 2006 Jul;39(3):189-98. doi: 10.1016/j.ymeth.2006.01.003.
Vectors based on murine retroviruses are among the most efficient means to insert reporter constructs into the context of a vertebrate chromosome with the aim to visualize cis-regulatory information available to a basal promoter at the site of insertion. In combination with using the zebrafish embryo as a readout for the activity of regulatory elements, enhancer detection becomes a powerful technique for gene discovery and for the mapping of the extent of regulatory domains in a vertebrate genome. Our laboratory has performed the only large-scale enhancer detection screen to date in any vertebrate and we describe in this paper the methods we developed to generate viral particles, to insert reporter constructs into the zebrafish germ line, the screening of detection events in heterozygous F1 embryos, and the isolation of genomic sequence flanking the inserted vector for the purpose of genomic mapping. Given sufficient scale, the technology described here can be used to obtain cis-regulatory information across the entire zebrafish genome for any given basal promoter.
基于鼠逆转录病毒的载体是将报告基因构建体插入脊椎动物染色体背景的最有效手段之一，目的是可视化插入位点处基础启动子可利用的顺式调控信息。结合使用斑马鱼胚胎作为调控元件活性的读出系统，增强子检测成为一种强大的技术，可用于基因发现以及绘制脊椎动物基因组中调控域的范围。我们实验室进行了迄今为止在任何脊椎动物中唯一的大规模增强子检测筛选，本文中我们描述了我们开发的用于产生病毒颗粒、将报告基因构建体插入斑马鱼生殖系、在杂合F1胚胎中筛选检测事件以及分离插入载体侧翼基因组序列以进行基因组定位的方法。如果规模足够大，这里描述的技术可用于获得任何给定基础启动子在整个斑马鱼基因组中的顺式调控信息。
