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{"title":"单细胞中端粒长度、端粒伸长和TERT表达的原位可视化","authors":"Ajay Ravindranathan, Morgan E. Diolaiti, Beth A. Cimini, Bradley A. Stohr","doi":"10.1002/cpcb.97","DOIUrl":null,"url":null,"abstract":"<p>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.</p><p><b>Basic Protocol 1</b>: TSQ1 lentivirus production</p><p><b>Basic Protocol 2</b>: TSQ1 lentiviral infection and plating</p><p><b>Basic Protocol 3</b>: RNAscope analysis</p><p><b>Basic Protocol 4</b>: TSQ1 PNA-FISH detection</p>","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"85 1","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2019-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.97","citationCount":"1","resultStr":"{\"title\":\"In Situ Visualization of Telomere Length, Telomere Elongation, and TERT Expression in Single Cells\",\"authors\":\"Ajay Ravindranathan, Morgan E. Diolaiti, Beth A. Cimini, Bradley A. Stohr\",\"doi\":\"10.1002/cpcb.97\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>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.</p><p><b>Basic Protocol 1</b>: TSQ1 lentivirus production</p><p><b>Basic Protocol 2</b>: TSQ1 lentiviral infection and plating</p><p><b>Basic Protocol 3</b>: RNAscope analysis</p><p><b>Basic Protocol 4</b>: TSQ1 PNA-FISH detection</p>\",\"PeriodicalId\":40051,\"journal\":{\"name\":\"Current Protocols in Cell Biology\",\"volume\":\"85 1\",\"pages\":\"\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2019-10-03\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://sci-hub-pdf.com/10.1002/cpcb.97\",\"citationCount\":\"1\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Current Protocols in Cell Biology\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1002/cpcb.97\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"Biochemistry, Genetics and Molecular Biology\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current Protocols in Cell Biology","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cpcb.97","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
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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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