Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome
Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome
复制标题
DOI：
10.1038/ng1312
复制
发表时间：
2004-03-01
期刊：
影响因子：
30.8
通讯作者：
Francis-Lang, HL
中科院分区：
文献类型：
--
作者：
Parks, AL;Cook, KR;Francis-Lang, HL
在果蝇研究中，染色体缺失是绘制突变图谱、表征等位基因以及鉴定相互作用位点不可或缺的工具。最广泛使用的缺失是通过辐射或化学诱变产生的。这些方法劳动强度大，产生随机断点，并导致不需要的二次突变，这会使表型分析变得复杂。大多数现有的缺失片段较大，端点在分子层面未明确界定，并且保存在遗传复杂的种群中。此外，单倍致死或单倍不育位点的存在使得通过传统方法恢复某些区域的缺失极其困难，从而导致覆盖范围存在缺口。在此我们描述了两种方法，通过在黑腹果蝇基因组中系统地分离靶向缺失来解决这些问题。第一种策略使用基于P元件的技术来产生紧密环绕单倍不足基因的缺失，并使未缺失区域最小化。这个缺失组使整体基因组覆盖度提高了5 - 7%。第二种策略使用FLP重组酶以及在相关论文（1）中描述的大量带有FRT的插入来产生519个端点在分子层面明确界定的同基因缺失。到目前为止，第二个缺失集合提供了56%的基因组覆盖度。后一种方法能够在整个基因组中产生具有可预测端点的小型定制缺失，并应使其分离成为一项简单的常规任务。
In fruit fly research, chromosomal deletions are indispensable tools for mapping mutations, characterizing alleles and identifying interacting loci. Most widely used deletions were generated by irradiation or chemical mutagenesis. These methods are labor-intensive, generate random breakpoints and result in unwanted secondary mutations that can confound phenotypic analyses. Most of the existing deletions are large, have molecularly undefined endpoints and are maintained in genetically complex stocks. Furthermore, the existence of haplolethal or haplosterile loci makes the recovery of deletions of certain regions exceedingly difficult by traditional methods, resulting in gaps in coverage. Here we describe two methods that address these problems by providing for the systematic isolation of targeted deletions in the D. melanogaster genome. The first strategy used a P element based technique to generate deletions that closely flank haploinsufficient genes and minimize undeleted regions. This deletion set has increased overall genomic coverage by 5-7%. The second strategy used FLP recombinase and the large array of FRT-bearing insertions described in the accompanying paper(1) to generate 519 isogenic deletions with molecularly defined endpoints. This second deletion collection provides 56% genome coverage so far. The latter methodology enables the generation of small custom deletions with predictable endpoints throughout the genome and should make their isolation a simple and routine task.