Stable-isotope Labeled Metabolic Analysis in Drosophila melanogaster: From Experimental Setup to Data Analysis
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Author:
Date:
2018-09-20
[Abstract] Stable-isotope labeled metabolic analysis is an essential methodology to characterize metabolic regulation during biological processes. However, the method using stable-isotope-labeled tracer (e.g., 13C-glucose) in live animal is only beginning to be developed. Here, we contribute a qualitative metabolic labeling experiment protocol in Drosophila melanogaster using stable-isotope-labeled 13C-glucose tracer followed by liquid chromatography-mass spectrometry (LC-MS) analysis. Detailed experimental setup, data acquisition and analysis are provided to facilitate the application of in vivo metabolic labeling analysis that might be applied in a wide range of biological studies.
[摘要] 稳定同位素标记的代谢分析是表征生物过程中代谢调节的基本方法。 然而,在活体动物中使用稳定同位素标记的示踪剂(例如, 13 C-葡萄糖)的方法才刚刚开始开发。 在这里,我们使用稳定同位素标记的 13 C-葡萄糖示踪剂,然后通过液相色谱 - 质谱(LC-MS)在 Drosophila melanogaster 中提供定性代谢标记实验方案。 分析。 提供详细的实验设置,数据采集和分析以促进体内代谢标记分析的应用,其可以应用于广泛的生物学研究中。
【背景】代谢组学是一项新兴的omic-level研究,旨在研究复杂生物系统中的小分子代谢物。它已被应用于与人类健康和疾病有关的各种研究领域,例如生物标志物发现,疾病发病机理和药物毒性评估。代谢物的测量对于确定响应内源和外源变化的代谢途径的改变是重要的。为了准确表征代谢途径活动,已使用同位素标记的示踪剂(例如, 13 C和 15 N)(Park et al。,2016; Jang et al。,2018)。在培养的细胞中有许多这样的研究(定量和定性)(Buescher et al。,2015; Liu et al。,2018),然而,基于稳定同位素的研究活体动物的代谢标记实验在很大程度上尚未开发。在目前的方案中,我们描述了使用标记的 13 ...
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Arabidopsis-Green Peach Aphid Interaction: Rearing the Insect, No-choice and Fecundity Assays, and Electrical Penetration Graph Technique to Study Insect Feeding Behavior
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Author:
Date:
2018-08-05
[Abstract] Aphids constitute a large group of Hemipterans that use their slender stylets to tap into the sieve elements of plants from which they consume copious amounts of phloem sap, thus depriving the plant of photoassimilates. Some aphids also transmit viral diseases of plants. Myzus persicae Sülzer, commonly known as the green peach aphid (GPA), which is a polyphagous insect with a host range that covers 50 plant families, is considered amongst the top 3 insect pest of plants. The interaction between Arabidopsis thaliana and the GPA is utilized as a model pathosystem to study plant-aphid interaction. Here we describe the protocol used in our laboratories for rearing the GPA, and no-choice and fecundity bioassays to study GPA performance on Arabidopsis. In addition, we ...
[摘要] 蚜虫构成了一大群半翅目,它们使用细长的探针进入植物的筛子元素,从而消耗大量的韧皮部汁液,从而剥夺了植物的光合同化物。 一些蚜虫也传播植物的病毒性疾病。 桃蚜(Myzus persicae)Sülzer,俗称绿桃蚜(GPA),是一种多食性昆虫,寄主范围覆盖50个植物科,被认为是植物的三大害虫之一。 拟南芥与GPA之间的相互作用被用作研究植物 - 蚜虫相互作用的模型病理系统。 在这里,我们描述了我们的实验室用于饲养GPA的方案,以及用于研究拟南芥的GPA性能的无选择和繁殖生物测定。 此外,我们描述了电穿透图(EPG)技术的程序,以监测GPA在拟南芥上的摄食行为。
【背景】蚜虫是植物的重要害虫,它们利用它们的口器被改造成管心针,以从筛子元件中去除韧皮部汁液。作为其饲养过程的一部分,蚜虫将唾液沉积到植物组织中。虽然一些唾液成分引发植物防御,但其他驯鹿成分则操纵宿主生理学以使昆虫受益,包括抑制植物防御(Nalam et al。,2018)。植物利用各种防御来控制蚜虫侵扰。这些包括抗生素,它对蚜虫的生长,发育和繁殖以及影响昆虫行为(包括摄食行为)的抗病毒产生不利影响。当探针尖端在植物细胞内时,以及当探针尖端在筛子元件中时,这些防御在各个步骤施加,包括在细胞表面,在昆虫探针的叶组织细胞间穿透期间(Nalam 等人,,2018)。绿桃蚜(GPA), Myzus ...
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Plasmid Extract from Budding Yeast (Saccharomyces cerevisiae)
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Author:
Date:
2018-07-20
[Abstract] Plasmids are widely used tools in yeast research. In many cases, plasmid libraries are used in genetic screens or in yeast two hybrid screens. In such cases, it is necessary to extract plasmids carrying unknown genetic elements from positive clones that were isolated in the screen.
This is a simple protocol to extract plasmid DNA from budding yeast cultures (Robzyk and Kassir, 1992). The amount produced is small, but it is sufficient for PCR or for transformation into bacteria, where the plasmid can be amplified to provide sufficient amounts for downstream uses (e.g., restriction enzyme analysis, sequencing).
[摘要] 质粒是酵母研究中广泛使用的工具。 在许多情况下,质粒文库用于遗传筛选或酵母双杂交筛选。 在这种情况下,有必要从筛选中分离的阳性克隆中提取携带未知遗传元件的质粒。
这是从芽殖酵母培养物中提取质粒DNA的简单方案(Robzyk和Kassir,1992)。 产生的量很小,但它足以用于PCR或转化到细菌中,其中质粒可以被扩增以提供足够的量用于下游用途(例如,,限制酶分析,测序)。
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