Detection of hTERT protein by flow cytometry
Detection of hTERT protein by flow cytometry
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DOI：
10.1038/sj.leu.2401950
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发表时间：
2000-12-01
期刊：
影响因子：
11.4
通讯作者：
Testa, NG
中科院分区：
文献类型：
--
作者：
Ali, ASM;Chopra, R;Testa, NG
端粒酶是一种端粒特异的DNA聚合酶，由蛋白质和RNA组成，在生殖细胞和大多数癌症中被激活，用于对抗端粒缩短的后果。HTERT被认为是人类端粒酶的催化亚单位，其在mRNA水平的表达与体外端粒酶活性密切相关。目前检测端粒酶活性的技术只能检测样本中的平均活性，不能分离特定的细胞亚群。这份报告描述了一种基于免疫荧光的细胞流式细胞术的开发和验证，该方法可以在保持可识别的细胞群体特征的同时检测核内hTERT。结果表明，该方法对端粒酶表达的变化很敏感，而且是半定量的。在细胞分化实验和原代细胞中，流式细胞仪检测的hTERT表达与端粒重复序列扩增法(TRAP)检测的端粒酶活性之间存在良好的相关性。建立的方法提供了一种快速、简单和可重复性的基于细胞的hTERT表达检测方法。本实验为今后的研究提供了一种有用的新工具，有助于分析hTERT在混合细胞群体中的表达。
Telomerase is a telomere-specific DNA polymerase consisting of protein and RNA components, which is activated in germline cells and the majority of cancers and serves to counter the consequences of telomere shortening. The protein component, hTERT, is believed to be the catalytic subunit of human telomerase and its expression at the mRNA level correlates well with telomerase activity in vitro. Current techniques for assaying telomerase activity detect only the mean activity in a sample and are unable to isolate specific cell sub-populations. This report describes the development and validation of a cellular, immunofluorescence-based flow cytometry assay that allows detection of intranuclear hTERT while maintaining identifiable cell population characteristics. The assay was shown to be both sensitive to changes in telomerase expression and was semi-quantitative. In both cell line differentiation experiments and in primary cells, a good correlation existed between hTERT expression measured by flow cytometry and telomerase activity detected by the telomeric repeat amplification protocol (TRAP). The method developed offers a quick, simple and reproducible cellular-based assay for hTERT expression. This assay will provide a useful, new tool for future investigations, facilitating the analysis of hTERT expression in mixed cell populations.