SIMPLE AND EFFICIENT GENERATION OF MARKED CLONES IN DROSOPHILA
SIMPLE AND EFFICIENT GENERATION OF MARKED CLONES IN DROSOPHILA
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DOI：
10.1016/0960-9822(93)90349-s
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发表时间：
1993-07-01
期刊：
影响因子：
9.2
通讯作者：
PERRIMON, N
中科院分区：
文献类型：
--
作者：
HARRISON, DA;PERRIMON, N
背景：细胞谱系分析和突变的嵌合体分析是用于研究许多生物体发育的重要技术。不幸的是，用于这种分析的方法通常效率低下，技术要求高或劳动密集型。在果蝇中，产生嵌合体动物最常用的方法是由X射线诱导的有丝分裂重组。尽管该技术简单，但其具有低效率和高细胞死亡率的不良特性。此外，虽然大量的标记系统已被用来检测有丝分裂重组体，没有允许容易识别的克隆为所有细胞types.Results：一个系统在这里描述，允许一个高效率的代克隆与伴随的表达一个容易检测的细胞标记。该方法可应用于果蝇的细胞谱系和嵌合体分析。在热激诱导型启动子的控制下，位点特异性酵母FLP重组酶有效地催化特异性在FLP重组靶位点（FRT）的有丝分裂重组。在这个系统中，重组融合α-微管蛋白启动子的lacZ基因，允许转录的标志物。因此，重组细胞及其后代可以通过β-半乳糖苷酶的标准测定法检测。特别重要的是，只有感兴趣的细胞染色的事实，从而允许他们简单的检测在任何tissue.Conclusions：我们证明，通过分子间重组，我们可以使用FLP重组酶产生标记克隆有效地在胚胎，幼虫和成人组织。这种简单而有效的技术非常适合细胞谱系分析，并可以很容易地扩展到嵌合体动物突变克隆的产生和检测。
Background: Cell lineage analysis and mosaic analysis of mutations are important techniques that are used to study the development of many organisms. Unfortunately, the methods employed for such analyses are usually inefficient, technically demanding or labor intensive. In Drosophila, the most common methodology used for the generation of mosaic animals is mitotic recombination, which is induced by X-rays. Although this technique is simple, it has the undesirable characteristics of a low efficiency and a high rate of cell death. Furthermore, although a large number of marker systems has been employed to detect mitotic recombinants, none allows easy identification of clones for all cell types.Results: A system is described here that allows a highly efficient generation of clones with the concomitant expression of an easily detectable cellular marker. This method can be applied to cell lineage and mosaic analysis in Drosophila The site-specific yeast FLP recombinase, under the control of a heat shock-inducible promoter, efficiently catalyses mitotic recombination specifically at the site of a FLP recombination target (FRT). In this system, recombination fuses the alpha-tubulin promoter to the lacZ gene, allowing transcription of the marker. Recombinant cells and their progeny can, therefore, be detected by standard assays for beta-galactosidase. of particular importance is the fact that only the cells of interest stain, thus allowing their simple detection in any tissue.Conclusions: We demonstrate that, by intermolecular recombination, we can use FLP recombinase to generate marked clones efficiently in embryonic, larval and adult tissues. This simple and efficient technique is well suited to cell-lineage analysis and can be easily extended to the generation and detection of mutant clones in mosaic animals.