单细胞中端粒长度、端粒伸长和TERT表达的原位可视化

Q3 Biochemistry, Genetics and Molecular Biology

Current Protocols in Cell Biology Pub Date : 2019-10-03 DOI:10.1002/cpcb.97

Ajay Ravindranathan, Morgan E. Diolaiti, Beth A. Cimini, Bradley A. Stohr

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引用次数: 1

摘要

端粒酶通过在端粒末端添加六核苷酸重复序列，延长细胞增殖寿命，在癌症和衰老中发挥关键作用。端粒酶在大多数细胞群中的表达水平极低，且单细胞端粒伸长检测困难，限制了对异质群体中单个细胞端粒酶表达和功能的研究。本文描述的方法将端粒酶逆转录酶(TERT)的单分子检测(RNAscope)与我们之前描述的用于端粒延伸原位监测的TSQ1检测相结合，从而能够检测单细胞中的TERT表达、端粒长度和端粒伸长，并为研究端粒酶调节端粒伸长的因素提供了一种独特的方法。©2019 by John Wiley &基本协议1:TSQ1慢病毒生产基本协议2:TSQ1慢病毒感染和镀皿基本协议3:RNAscope分析基本协议4:TSQ1 PNA-FISH检测

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In Situ Visualization of Telomere Length, Telomere Elongation, and TERT Expression in Single Cells

Telomerase plays a critical role in cancer and aging by adding hexa-nucleotide repeats to the ends of telomeres and extending the cellular proliferative lifespan. The very low level of telomerase expression in most cell populations and the difficulty of detecting telomere elongation in single cells have limited the study of telomerase expression and function in individual cells of a heterogeneous population. The method described in this article combines single-molecule detection (RNAscope) of telomerase reverse transcriptase (TERT) with our previously described TSQ1 assay for in situ monitoring of telomere extension, thereby enabling detection of TERT expression, telomere length, and telomere elongation in single cells and providing a unique approach for studying the factors that regulate telomere elongation by telomerase. © 2019 by John Wiley & Sons, Inc.

Basic Protocol 1: TSQ1 lentivirus production

Basic Protocol 2: TSQ1 lentiviral infection and plating

Basic Protocol 3: RNAscope analysis

Basic Protocol 4: TSQ1 PNA-FISH detection

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Current Protocols in Cell Biology Biochemistry, Genetics and Molecular Biology-Cell Biology

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期刊介绍： Developed by leading scientists in the field, Current Protocols in Cell Biology is an essential reference for researchers who study the relationship between specific molecules and genes and their location, function and structure at the cellular level. Updated every three months in all formats, CPCB is constantly evolving to keep pace with the very latest discoveries and developments.