Simple methods for generating and detecting locus-specific mutations induced with TALENs in the zebrafish genome.
Simple methods for generating and detecting locus-specific mutations induced with TALENs in the zebrafish genome.
复制标题
DOI：
10.1371/journal.pgen.1002861
复制
发表时间：
2012
期刊：
影响因子：
4.5
通讯作者：
Grunwald DJ
中科院分区：
文献类型：
--
作者：
Dahlem TJ;Hoshijima K;Jurynec MJ;Gunther D;Starker CG;Locke AS;Weis AM;Voytas DF;Grunwald DJ
斑马鱼是揭示脊椎动物基因功能的强大实验系统。然而，斑马鱼的研究受到可用于消除基因功能的方法的限制。在这里，我们提出了简单有效的方法来诱导、检测和恢复斑马鱼几乎任何位点的突变。简而言之，合成核酸酶（称为 TALEN）在目标位点诱导双链 DNA 断裂。随后宿主对 DNA 损伤的修复导致目标位点产生插入和缺失突变。为了检测目标位点处诱导的 DNA 序列改变，使用高分辨率熔解分析来检查基因组，这是一种有效且灵敏的方法，用于检测新出现的序列多态性的存在。由于 TALEN 的 DNA 结合特异性是由定制设计的 DNA 识别模块阵列决定的，每个模块都与单个靶核苷酸相互作用，因此可以轻松生成具有非常高靶序列特异性的 TALEN。使用可免费获取的试剂和基于网络的软件以及非常简单的克隆策略，可以在几天内生成能够唯一识别斑马鱼基因组中特定预定位点的 TALEN。在这里，我们开发并测试了四种针对不同靶基因的 TALEN 的活性。使用此处描述的实验方法，每个注射了编码 TALEN 的 RNA 的胚胎都会获得靶向突变。每个生长的胚胎中都会产生多个独立发生的突变，高达 50% 的宿主基因组可能会获得目标突变。成年后，大约 90% 的动物会将靶向突变遗传给它们的后代。这里给出的结果表明 TALEN 具有高度序列特异性，并且产生最小的脱靶效应。总而言之，创建目标特异性 TALEN 并生成在预先指定的基因座上包含一系列种系突变的生长胚胎需要大约两周的时间。许多基因的发现仅仅是基于它们与某种性状或疾病的关联，或者它们与其他已知基因的相关性，但尽管如此，这些基因的精确生物学功能仍然是个谜。我们需要新的工具来发现感兴趣基因的直接分子、细胞和发育功能。斑马鱼越来越多地被用作模式生物来发现所有脊椎动物共有的基因功能。在这项研究中，我们开发了新的、高效且非常容易应用的方法来生成缺乏任何所需基因功能的斑马鱼。我们还介绍了灵敏且易于应用的方法来检测新出现的突变。这里开发的方法还可用于快速消除其他动物或组织培养细胞中任何选定基因的功能。总之，我们预计这里描述的方法将广泛应用于许多不同背景下的基因功能研究。
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 target sequence specificities can be easily generated. Using freely accessible reagents and Web-based software, and a very simple cloning strategy, a TALEN that uniquely recognizes a specific pre-determined locus in the zebrafish genome can be generated within days. Here we develop and test the activity of four TALENs directed at different target genes. Using the experimental approach described here, every embryo injected with RNA encoding a TALEN will acquire targeted mutations. Multiple independently arising mutations are produced in each growing embryo, and up to 50% of the host genomes may acquire a targeted mutation. Upon reaching adulthood, approximately 90% of these animals transmit targeted mutations to their progeny. Results presented here indicate the TALENs are highly sequence-specific and produce minimal off-target effects. In all, it takes about two weeks to create a target-specific TALEN and generate growing embryos that harbor an array of germ line mutations at a pre-specified locus. Many genes are being discovered solely on the basis of their association with a trait or disease, or their relatedness to other known genes, but nevertheless the precise biological functions of these genes remain mysterious. We need new tools to discover the immediate molecular, cellular, and developmental functions of genes of interest. Increasingly, the zebrafish is being used as a model organism to discover gene functions that are shared among all vertebrates. In this study we develop new, highly efficient, and very easy to apply methods for generating zebrafish that lack the function of any desired gene. We also introduce sensitive and easy-to-apply methods for detecting newly arising mutations. The approach developed here can also be used to quickly eliminate the function of any chosen gene in other animals or in tissue culture cells. In all, we anticipate the methods described here will be widely applied to study gene function in many different contexts.
登录
查看更多内容
影响因子：
56.9
作者：
Boch, Jens;Scholze, Heidi;Bonas, Ulla
通讯作者：
Bonas, Ulla
影响因子：
1.5
作者：
Draper, BW;Morcos, PA;Kimmel, CB
通讯作者：
Kimmel, CB
影响因子：
2.5
作者：
KIMMEL, CB;BALLARD, WW;SCHILLING, TF
通讯作者：
SCHILLING, TF
人类和斑马鱼肌肉中兰尼碱受体钙释放通道活性需要硒蛋白 N
DOI：
10.1073/pnas.0806015105
发表时间：
2008-08-26
影响因子：
11.1
作者：
Jurynec, Michael J.;Xia, Ruohong;Grunwald, David Jonah
通讯作者：
Grunwald, David Jonah
影响因子：
46.9
作者：
Miller, Jeffrey C.;Tan, Siyuan;Rebar, Edward J.
通讯作者：
Rebar, Edward J.