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Proceedings Paper

Quantification of telomere length by FISH and laser scanning cytometry
Author(s): John E. Mahoney; Ergun Sahin; Mariela Jaskelioff; Lynda Chin; Ronald A. DePinho; Alexei I. Protopopov
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Paper Abstract

Telomeres play a critical role in the maintenance of chromosomal stability. Telomere erosion, coupled with loss of DNA damage checkpoint function, results in genomic instability that promotes the development of cancer. The critical role of telomere dynamics in cancer has motivated the development of technologies designed to monitor telomere reserves in a highly quantitative and high-throughput manner in humans and model organisms. To this end, we have adapted and modified two established technologies, telomere-FISH and laser scanning cytometry. Specifically, we have produced a number of enhancements to the iCys LSC (CompuCyte) package including software updates, use of 60X dry objectives, and increased spatial resolution by 0.2 um size of stage steps. In addition, the 633 nm HeNe laser was replaced with a 532 nm green diode laser to better match the viewing options. Utilization of telomere-deficient mouse cells with short dysfunctional telomeres and matched telomerase reconstituted cultures demonstrated significantly higher mean integral specific fluorescence values for mTR transfectants relative to empty vector controls: 4.485M vs. 1.362M (p<0.0001). Histograms of average telomere intensities for individual cells were obtained and demonstrated intercellular heterogeneity in telomere lengths. The validation of the approach derives from a strong correlation between iCys LSC values and Southern blotting. This validated method greatly increases our experimental throughput and objectivity.

Paper Details

Date Published: 29 February 2008
PDF: 9 pages
Proc. SPIE 6859, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues VI, 68591V (29 February 2008); doi: 10.1117/12.763858
Show Author Affiliations
John E. Mahoney, Harvard Medical School (United States)
Ergun Sahin, Harvard Medical School (United States)
Mariela Jaskelioff, Harvard Medical School (United States)
Lynda Chin, Harvard Medical School (United States)
Ronald A. DePinho, Harvard Medical School (United States)
Alexei I. Protopopov, Harvard Medical School (United States)

Published in SPIE Proceedings Vol. 6859:
Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues VI
Daniel L. Farkas; Dan V. Nicolau; Robert C. Leif, Editor(s)

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