Multi-allelic phenotyping - A systematic approach for the simultaneous analysis of multiple induced mutations
Multi-allelic phenotyping - A systematic approach for the simultaneous analysis of multiple induced mutations
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DOI：
10.1016/j.ymeth.2013.04.013
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发表时间：
2013-08-15
期刊：
影响因子：
4.8
通讯作者：
Busch-Nentwich, Elisabeth M.
中科院分区：
文献类型：
--
作者：
Dooley, Christopher M.;Scahill, Catherine;Busch-Nentwich, Elisabeth M.
斑马鱼突变项目（ZMP）的目标是为每个蛋白质编码基因产生一个功能丧失的等位基因，但重要的是在发育的前五天也要改变这些等位基因的表型。这样一个大规模的屏幕需要一个系统的方法来识别表型，也将这些表型与特定的突变。该表型分析管道通过两步过程同时评估多个等位基因的后果。首先，鉴定在发育的前五天内不产生可见表型的突变，而第二轮表型分析侧重于对怀疑引起表型的等位基因进行详细分析。等位基因特异性PCR单核苷酸多态性（SNP）分析用于基因型F2父母和个别F3鱼苗的突变已知是存在于F1的创始人。通过这种方法，可以将特定的表型与诱导突变联系起来。此外，还描述了一种冷冻保存诱变雄性精子样品的方法，并随后将其用于体外受精以产生用于表型分析的F2家族。最终，这种方法将导致斑马鱼基因组的功能注释，这将加深我们对发育和疾病中基因功能的理解。(C)2013作者爱思唯尔公司出版All rights reserved.
The zebrafish mutation project (ZMP) aims to generate a loss of function allele for every protein-coding gene, but importantly to also characterise the phenotypes of these alleles during the first five days of development. Such a large-scale screen requires a systematic approach both to identifying phenotypes, and also to linking those phenotypes to specific mutations. This phenotyping pipeline simultaneously assesses the consequences of multiple alleles in a two-step process. First, mutations that do not produce a visible phenotype during the first five days of development are identified, while a second round of phenotyping focuses on detailed analysis of those alleles that are suspected to cause a phenotype. Allele-specific PCR single nucleotide polymorphism (SNP) assays are used to genotype F2 parents and individual F3 fry for mutations known to be present in the F1 founder. With this method specific phenotypes can be linked to induced mutations. In addition a method is described for cryopreserving sperm samples of mutagenised males and their subsequent use for in vitro fertilisation to generate F2 families for phenotyping. Ultimately this approach will lead to the functional annotation of the zebrafish genome, which will deepen our understanding of gene function in development and disease. (C) 2013 The Authors. Published by Elsevier Inc. All rights reserved.