Target-selected mutant screen by TILLING in Drosophila
Target-selected mutant screen by TILLING in Drosophila
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DOI：
10.1101/gr.3721805
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发表时间：
2005-05-01
期刊：
影响因子：
7
通讯作者：
González-Gaitán, M
中科院分区：
文献类型：
--
作者：
Winkler, S;Schwabedissen, A;González-Gaitán, M
完整的果蝇基因组DNA序列的可用性促进了一种方法的发展，以有效地获得通过序列同源性或生物化学鉴定的感兴趣基因突变。迄今为止，分子特征突变已经在-15,000个注释果蝇基因中的约6000个中产生，其中约三分之一是生存所必需的。为了获得必要和非必要基因的突变，我们采用了一种反向遗传学方法，基于大规模检测细胞- i介导的异双工切割的点突变。建立了2086个ems诱变品系的基因组DNA文库。在文库中筛选了三个基因的突变。总共筛选了6.1 Mb，在两种不同的诱变条件下发现了44个hit。在最佳条件下，平均每156 kb产生一个突变。在所有的基本基因测试中，25个突变中有5个被证明是致命的，这证实了EMS突变导致基因失活的频率很高。因此，我们确定了细胞i介导的TILLING呼叫可用于有效地获得果蝇感兴趣的基因突变。
The availability of the full Drosophila genomic DNA sequence prompts the development of a method to efficiently obtain mutations in genes of interest identified by their sequence homologies or biochemically. To date, molecularly characterized Mutations have been generated in around 6000 of the -15,000 annotated fly genes, of which around one-third are essential for viability. To obtain Mutations in essential and nonessential genes of interest, we took a reverse genetics approach, based oil the large-scale detection Of point Mutations by Cel-I-mediated heteroduplex cleavage. A library of genomic DNA from 2086 EMS-mutagenized lines was established. The library was screened for mutations ill three genes. A total of 6.1 Mb were screened, and 44 hits were found in two different mutagenesis conditions. Optimal conditions yielded ail average of one Mutation every 156 kb. For ail essential gene tested, five of 25 Mutations turned Out to cause lethality, confirming that EMS mutagenesis leads to high frequency of gene inactivation. We thereby established that Cel-I-mediated TILLING call be Used to efficiently obtain mutations in genes of interest in Drosophila.