The large-scale Munich ENU-mouse-mutagenesis screen
The large-scale Munich ENU-mouse-mutagenesis screen
复制标题
大规模慕尼黑 ENU 小鼠诱变筛选
DOI：
10.1007/s003350010097
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发表时间：
2000
期刊：
影响因子：
2.5
通讯作者：
M. Hrabě de Angelis
中科院分区：
文献类型：
--
作者：
D. Soewarto;C. Fella;A. Teubner;B. Rathkolb;W. Pargent;S. Heffner;S. Marschall;E. Wolf;R. Balling;M. Hrabě de Angelis
在接下来的几年内，人类基因组的完整序列将可用(Schuler等人。1996)，后基因组时代将开始对基因功能及其在人类发病和疾病中的作用进行系统分析。自发突变和诱导突变的特征、转基因和基因靶向动物(如果蝇、斑马鱼或啮齿动物)的表型分析，是深入了解基因生物学功能的常用工具。关于人类疾病的遗传学和发病机制，动物模型对于进一步研究是必不可少的；特别是小鼠，由于其基因组、发育和生化途径以及生理学与人类的相似性，作为模型系统发挥了重要作用。可以尝试两种不同的策略来系统地在小鼠中产生突变表型：基因驱动和表型驱动的方法。基因驱动的方法基于小鼠胚胎干细胞技术，在该技术中，可以为通过同源重组而设计的任何定向突变产生小鼠突变(Thomas和Capecchi，1987；Ramirez-Solis等人)。1993年)。限制因素是靶向构建体的产生，该构建体涉及包含所需突变的工程DNA片段。到目前为止，由于基因结构的异质性，这项技术的大规模自动化还无法进行。在大多数情况下，工程构建取代了野生型基因的目标DNA区域，并中断了基因的功能，从而导致等位基因无效或基因被敲除。另一种基因驱动的方法是基因陷阱策略(Evans等人。1997年)。在这种方法中，将一个可选择的插入片段引入ES细胞DNA，并通过建立大量携带被破坏单位的细胞系，建立用于产生小鼠突变体的ES细胞突变库(Wiles等人。2000)。基因驱动策略的缺点是产生的大多是零等位基因，通常不能揭示基因的所有生物学功能。对于人类临床上相关的疾病，人们关注的是部分但不是完全丧失基因功能的结果。因此，对于遗传性疾病的遗传分析，有必要产生单个基因的多个等位基因，这会导致亚型、不同强度的等位基因或功能获得等位基因。与这种基因驱动的方法相补充的是，在这种方法中，为那些已知的基因产生突变，表型驱动的方法通过恢复具有新表型的小鼠突变来识别新的基因、基因产物和它们相关的生物途径(Brown和Peter 1996)。电离辐射的随机诱变诱导大的缺失，与ENU(乙基亚硝胺)的化学诱变(Russell等人)。1979年，1990年；罗素1982；彼得斯1985；多芬1987；博德等人。1988年；Favy et al.1990a，1990b；Rinchik 1991)，导致点突变(Popp等人。1983年；哈巴赫等人。1992年)，而百菌清则会引起少量的缺失。这些方法在经典遗传学中有着悠久的传统，并产生了大量的突变表型。然后，通过位置克隆或其他策略确定负责的基因。这些方法的有效性和成功在对建立果蝇身体模式的途径的遗传和分子解剖过程中得到了证明(Nüsslein-Vollhard和Wieschaus 1980；Ashburner 1989)。这种表型驱动策略的主要兴趣是建立适当的程序来评估感兴趣的突变表型，获得人类疾病的动物模型，并深入了解基因功能。到目前为止，只有少数议定书满足了这些苛刻的要求(Rogers等人)。1997年)。德国人类基因组计划的ENU-小鼠突变项目(Hrabéde Angelis和Balling 1998)在过去3年中建立了病理生理异常的筛查和表型鉴定方案-慕尼黑方案，以评估特定的出生后异常的突变表型，包括先天性畸形、临床化学、生化、血液、免疫缺陷和复杂的特征，如过敏和行为。
Within the next few years the complete sequence of the human genome will be available (Schuler et al. 1996), and the postgenome era will start with the systematic analysis of gene function and its role in human pathogenesis and disease. Characterization of spontaneous and induced mutants, the analysis of transgenic and gene-targeted phenotypes in animals, i.e., fruitfly, zebrafish, or rodents, are common tools to obtain insight into the biological function of genes. With respect to the genetics and pathogenesis of human diseases, animal models are essential for further investigations; and in particular, the mouse has had a major role as a model system owing to the similarity of its genome, developmental and biochemical pathways, and physiology to humans. Two different strategies can be attempted for the systematic production of mutant phenotypes in the mouse: the gene-driven and the phenotype-driven approach. The gene-driven approach is based on the mouse embryonic stem cell technology, in which mouse mutants can be generated for any targeted mutation engineered through homologous recombination (Thomas and Capecchi 1987; Ramirez-Solis et al. 1993). The limiting factor is the production of the targeting construct, which involves an engineered DNA fragment containing the desired mutation. To date, automation of this technique on a large scale could not be carried out because of the heterogeneity of gene structures. In most cases, the engineered construct replaces the targeted DNA region of the wildtype gene and interrupts the gene’s function, which results in a null allele or a ‘knocked-out’ gene. Another gene-driven approach is the gene trap strategy (Evans et al. 1997). In this approach, a selectable insert is introduced into the ES cell DNA, and by building a large number of cell lines carrying disrupted units, an ES cell mutation bank is established for generating mouse mutants (Wiles et al. 2000). The disadvantage of the gene-driven strategy is the production of mostly null alleles, which often do not reveal all biological functions of a gene. With respect to clinically relevant diseases in humans, the focus is on the result of a partial, but not complete loss of gene function. Thus, for a genetic analysis of inherited diseases, it is necessary to generate multiple alleles of a single gene, which results in hypomorphs, alleles of different strength or gain-of-function alleles. Complementary to such a gene-driven approach, in which mutants are produced for those genes that are already known, the phenotype-driven approach identifies new genes, gene products, and their relevant biological pathways by recovering mouse mutants with a novel phenotype (Brown and Peters 1996). Random mutagenesis from ionizing radiation induces large deletions, chemical mutagenesis with ENU (ethylnitrosourea) (Russell et al. 1979, 1990; Russell 1982; Peters 1985; Dove 1987; Bode et al. 1988; Favor et al. 1990a, 1990b; Rinchik 1991), causes point mutations (Popp et al. 1983; Harbach et al. 1992), while chlorambucil induces small deletions. These approaches have a long tradition in classical genetics and have generated a large number of mutant phenotypes. The responsible genes are then identified through positional cloning or other strategies. The validity and success of these approaches were demonstrated in the course of the genetic and molecular dissection of the pathway that set up the Drosophila body pattern (Nüsslein-Vollhard and Wieschaus 1980; Ashburner 1989). The main interest in such a phenotype-driven strategy is the establishment of appropriate procedures to assess the mutant phenotypes of interest, to obtain animal models of human diseases and insight into the gene functions. To date, only a few protocols have met these demanding requirements (Rogers et al. 1997). A screening and phenotyping protocol for pathophysiological abnormalities—the Munich protocol—has been established in the last 3 years in the ENU-mouse-mutagenesis project of the German Human Genome Project (Hrabé de Angelis and Balling 1998) to assess mutant phenotypes for specific, postnatal abnormalities comprising congenital malformations, clinical chemical, biochemical, hematological, immunological defects and complex traits such as allergies and behavior.
影响斑马鱼胚胎发生的突变的遗传筛查。
DOI：
10.1242/dev.123.1.37
发表时间：
1996
期刊：
Development (Cambridge, England)
影响因子：
--
作者：
Driever,W;Solnica-Krezel,L;Schier,AF;Neuhauss,SC;Malicki,J;Stemple,DL;Stainier,DY;Zwartkruis,F;Abdelilah,S;Rangini,Z;Belak,J;Boggs,C
通讯作者：
Boggs,C
从斑马鱼种系中有效恢复 ENU 诱导的突变。
影响因子：
3.3
作者：
Solnica-Krezel,L;Schier,AF;Driever,W
通讯作者：
Driever,W