翻译核糖体亲性纯化(TRAP),用于从体内内皮细胞中分离RNA
Summary
我们通过结合RNA纯化结合的增强绿色荧光蛋白(EGFP)的EC特异性遗传标记,直接在小鼠脑、肺和心脏组织中直接从小鼠脑、肺和心脏组织中从血管内皮细胞(EC)中纯化核糖体结合体mRNA的mRNA。.
Abstract
许多研究仅限于使用体外细胞测定和整组织,或从动物中分离特定细胞类型,通过qPCR和RNA测序对转录组和基因表达进行体外分析。对复杂组织和器官中特定细胞类型的全面转录和基因表达分析对于了解基因调控的细胞和分子机制及其与组织平衡和器官的关系至关重要功能。本文以动物肺血管内皮内皮直接在体内分离核糖体结合RNA的方法为例。将描述组织处理和RNA纯化的具体材料和程序,包括RNA质量和产量的评估以及动脉生成基因测定的实时qPCR。这种方法,称为翻译核糖体亲和纯化(TRAP)技术,可用于基因表达的表征和转录组分析某些细胞类型直接在体内任何特定类型的复杂组织。
Introduction
在哺乳动物大脑、心脏和肺等复杂组织中,细胞异质性水平高使从整个组织样本中提取的基因表达数据分析复杂化。为了观察体内特定细胞类型的基因表达图谱,最近开发了一种新方法,允许对任何基因定义的细胞类型的整个翻译的mRNA补充进行查询。这种方法被称为翻译核糖体亲和纯化(TRAP)技术1,2。当与基因操纵动物中其他血管生成相关基因时,它是研究内皮细胞生物学和血管生成的有用工具。
我们已经表明,血管生成PKD-1信号和血管生成基因CD36的转录对内皮细胞(EC)分化和功能血管生成3,4,5,6至关重要。为了确定基因转录和EC转导中血管生成和代谢信号的分子机制,我们根据TRAP技术1创建了具有特定删除血管生成基因的转基因TRAP小鼠。,2.此外,在我们的TRAP动物中,它们不仅在血管内皮瘤或CD36基因的全局缺失中缺乏pkd-1或cd36基因,而且增强的绿色荧光蛋白(EGFP)也基因标记到EC正在翻译核糖体。TRAP允许直接从靶向组织的血管内皮内,对核糖体结合的mRNA进行亲亲化纯化,从而能够分析基因表达和识别与EC分化相关的新转录体,血管生成直接在体内条件。我们已经成功地从这些基因工程动物的内皮内皮体中分离出核糖体结合的RNA。纯化RNA可用于在调节EC分化和功能方面进一步描述血管生成或动脉生成基因。该协议提供了一个分步指南,用于实现TRAP方法,用于直接在体内的EC中分离mRNA。
Protocol
Representative Results
Discussion
血管生成是一个复杂的多步骤过程,其中EC特异性血管原基因转录和表达在EC分化和血管生成重编程3,4中起着至关重要的作用。为了克服细胞多样性和结构复杂性的障碍,以更好地了解哺乳动物血管系统在体内分子水平上的功能,我们创建了EC特异性TRAP小鼠,并配以EC特异性cd36,EC特异性pkd-1缺乏或全球cd36缺乏,使用在Pu实验室2中生成的多功?…
Declarações
The authors have nothing to disclose.
Acknowledgements
任博士的工作得到了美国心脏协会(13SDG14800019)的支持;BR),安的希望基金会(FP00011709;BR)、美国癌症协会(86-004-26;MCW癌症中心至BR)和国家卫生研究院(HL136423;BR);乔丹·帕尔默得到2018年MCW CTSI 500明星实习计划的支持;P. Moran 得到 NHLBI (5T35 HL072483-34) 的机构研究培训资助。
Materials
| 2100 Electrophoresis Bioanalyzer with Nanochips and Picochips | Agilent | G2939AA, 5067-1511 & 5067-1513 | |
| Cell scrapers | Sarstedt | 83.1832 | |
| Homogenizers | Fisher Scientific | K8855100020 | |
| Magnet (Dynamag-2) | Invitrogen | 123-21D | Will depend on purification scale; samples in 1.5-mL tubes can be concentrated on a DynaMag-2 |
| Minicentrifuge | Fisher Scientific | 05-090-100 | |
| NanoDrop 2000C spectrophotometer | Thermo Scientific | ND-2000C | |
| Refrigerated centrifuge | Eppendorf | 5430R | with rotor for 1.5-mL microcentrifuge tubes |
| RNase-free 1.5mL microcentrifuge tubes | Applied Biosystems | AM12450 | |
| Rnase-free 50-mL conical tubes | Applied Biosystems | AM12501 | |
| RNase-free 1000-μl filter tips | Rainin | RT-1000F | |
| RNase-free 200-μl filter tips | Rainin | RT-200F | |
| RNase-free 20-μl filter tips | Rainin | RT-20F | |
| Rotor for homogenizers | Yamato | LT-400D | |
| Tube rotator, Labquake brand | Thermo Fisher | 13-687-12Q |
Referências
- Heiman, M., Kulicke, R., Fenster, R. J., Greengard, P., Heintz, N. Cell type-specific mRNA purification by translating ribosome affinity purification (TRAP). Nature Protocols. 9, 1282-1291 (2014).
- Zhou, P., et al. Interrogating translational efficiency and lineage-specific transcriptomes using ribosome affinity purification. Proceedings of the National Academy of Sciences of the United States of America. 110, 15395-15400 (2013).
- Best, B., Moran, P., Ren, B. VEGF/PKD-1 signaling mediates arteriogenic gene expression and angiogenic responses in reversible human microvascular endothelial cells with extended lifespan. Molecular and Cellular Biochemistry. 446, 199-207 (2018).
- Ren, B., et al. LPA/PKD-1-FoxO1 Signaling Axis Mediates Endothelial Cell CD36 Transcriptional Repression and Proangiogenic and Proarteriogenic Reprogramming. Arteriosclerosclerosis Thrombosis, Vascular Biology. 36, 1197-1208 (2016).
- Ren, B. Protein Kinase D1 Signaling in Angiogenic Gene Expression and VEGF-Mediated Angiogenesis. Frontiers in Cell and Developmental Biology. 4, 37 (2016).
- Ren, B. FoxO1 transcriptional activities in VEGF expression and beyond: a key regulator in functional angiogenesis?. Journal of Pathology. 245, 255-257 (2018).
- Hupe, M., Li, M. X., Gertow Gillner, K., Adams, R. H., Stenman, J. M. Evaluation of TRAP-sequencing technology with a versatile conditional mouse model. Nucleic Acids Research. 42, e14 (2014).
- Dong, L., et al. Diet-induced obesity links to ER positive breast cancer progression via LPA/PKD-1-CD36 signaling-mediated microvascular remodeling. Oncotarget. 8, 22550-22562 (2017).
- Ren, B., et al. ERK1/2-Akt1 crosstalk regulates arteriogenesis in mice and zebrafish. Journal of Clinical Investigation. 120, 1217-1228 (2010).
- Skuli, N., et al. Endothelial HIF-2alpha regulates murine pathological angiogenesis and revascularization processes. Journal of Clinical Investigation. 122, 1427-1443 (2012).
