In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining.
In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining.
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DOI：
10.3791/54516
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发表时间：
2016-10-15
期刊：
影响因子：
--
通讯作者：
Xu H
中科院分区：
文献类型：
--
作者：
Chen Z;Xu H
线粒体基因组完全通过母系遗传。了解卵母细胞及其基因组是如何增殖并通过雌性卵母细胞从一代传递到下一代的是至关重要的。由于遗传的易处理性，以及卵母细胞发育过程中优雅、有序的简单性，果蝇卵子发生已成为线粒体研究的一个非常宝贵的系统。采用EdU（5-乙炔基-2 ′-脱氧尿苷）标记法检测果蝇卵巢线粒体DNA（mtDNA）的复制。该方法上级于BrdU（5-溴-2 '-脱氧尿苷）标记方法，因为其允许良好的结构保存和有效的荧光染料穿透整个包埋组织。在这里，我们描述了一个详细的协议标记复制线粒体DNA在果蝇成年卵巢EdU。提供了一些技术解决方案，以提高卵巢的活力，在准备期间保持其健康，并确保高质量的成像。新合成的mtDNA在卵巢中的可视化不仅揭示了惊人的时间和空间模式的mtDNA复制通过卵子发生，但也允许在各种遗传和药理学扰动下的mtDNA复制的简单定量。
The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2´-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues. Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.
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DOI：
10.1038/nrg1606
发表时间：
2005-05
期刊：
Nature reviews. Genetics
影响因子：
--
作者：
通讯作者：
--
影响因子：
9.2
作者：
McClung, C;Hirsh, J
通讯作者：
Hirsh, J
影响因子：
3.6
作者：
Tornoe, CW;Christensen, C;Meldal, M
通讯作者：
Meldal, M
影响因子：
30.8
作者：
Hill, Jahda H.;Chen, Zhe;Xu, Hong
通讯作者：
Xu, Hong
影响因子：
5.4
作者：
Iborra, Francisco J.;Kimura, Hiroshi;Cook, Peter R.
通讯作者：
Cook, Peter R.