simple methods for generating and detecting locus-specific mutations induced with talens in the zebrafish genome简单的方法来生成和检测locus-specific突变诱导斑马鱼基因组的取得.pdfVIP

Simple Methods for Generating and Detecting Locus- Specific Mutations Induced with TALENs in the Zebrafish Genome 1. 1. 1 1 2 Timothy J. Dahlem , Kazuyuki Hoshijima , Michael J. Jurynec , Derrick Gunther , Colby G. Starker , 1 1 2 1 Alexandra S. Locke , Allison M. Weis , Daniel F. Voytas , David Jonah Grunwald * 1 Department of Human Genetics, University of Utah, Salt Lake City, Utah, United States of America, 2 Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota, United States of America Abstract The zebrafish is a powerful experimental system for uncovering gene function in vertebrate organisms. Nevertheless, studies in the zebrafish have been limited by the approaches available for eliminating gene function. Here we present simple and efficient methods for inducing, detecting, and recovering mutations at virtually any locus in the zebrafish. Briefly, double-strand DNA breaks are induced at a locus of interest by synthetic nucleases, called TALENs. Subsequent host repair of the DNA lesions leads to the generation of insertion and deletion mutations at the targeted locus. To detect the induced DNA sequence alterations at targeted loci, genomes are examined using High Resolution Melt Analysis, an efficient and sensitive method for detecting the presence of newly arising sequence polymorphisms. As the DNA binding specificity of a TALEN is determined by a custom designed array of DNA recognition modules, each of which interacts with a single target nucleotide, TALENs with very high targ