Monitoring gene expression in a single Xenopus oocyte using multiple cytoplasmic collections and quantitative RT-PCR
Monitoring gene expression in a single Xenopus oocyte using multiple cytoplasmic collections and quantitative RT-PCR
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使用多个细胞质收集和定量 RT-PCR 监测单个爪蟾卵母细胞的基因表达
DOI：
10.1111/febs.12576
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发表时间：
2014
期刊：
影响因子：
5.4
通讯作者：
Fukami Y.
中科院分区：
文献类型：
--
作者：
1.Tokmakov AA;Hashimoto T;Hasegawa Y;Iguchi S;Iwasaki T;Fukami Y.
非洲爪蛙的卵母细胞和卵通常用于基因表达研究。然而，监测个体活卵母细胞中的转录水平仍然具有挑战性。为了应对这一挑战，我们使用了一种技术，该技术基于从单个卵母细胞中多次重复收集纳升体积的细胞质材料。用定量RT-PCR方法进行转录定量。这项技术可以监测单个卵母细胞中异源基因的表达，而不会影响其生存能力。我们还使用这种方法对活的Xenopus卵母细胞内源基因的表达进行了分析。尽管青蛙卵母细胞传统上被认为是一个同质的细胞群体，但在个体卵母细胞中观察到了显著程度的基因表达差异。在卵母细胞群体中，转录水平呈对数正态分布。最后，利用这项技术，我们观察到在老化的未受精卵中，各种细胞质mRNAs的含量急剧下降，但在卵母细胞中却没有，这表明mRNA降解与卵细胞凋亡之间存在联系。
Oocytes and eggs of the African clawed frog,Xenopus laevis, are commonly used in gene expression studies. However, monitoring transcript levels in the individual living oocytes remains challenging. To address this challenge, we used a technique based on multiple repeated collections of nanoliter volumes of cytoplasmic material from a single oocyte. Transcript quantification was performed by quantitative RT‐PCR. The technique allowed monitoring of heterologous gene expression in a single oocyte without affecting its viability. We also used this approach to profile the expression of endogenous genes in livingXenopusoocytes. Although frog oocytes are traditionally viewed as a homogenous cell population, a significant degree of gene expression variation was observed among the individual oocytes. A lognormal distribution of transcript levels was revealed in the oocyte population. Finally, using this technique, we observed a dramatic decrease in the content of various cytoplasmic mRNAs in aging unfertilized eggs but not in oocytes, suggesting a link between mRNA degradation and egg apoptosis.