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Biotechnol J. 2012 Jan;7(1):103-16. doi: 10.1002/biot.201000474. Epub 2011 Jun 7.

A three-dimensional colocalization RNA interference screening platform to elucidate the alternative lengthening of telomeres pathway.

Author information

1
Research Group Genome Organization and Function, DKFZ and BioQuant, Heidelberg, Germany.

Abstract

A high-content colocalization RNA interference screen based on automatic three-color confocal fluorescence microscopy was developed to analyze the alternative lengthening of telomeres (ALT) pathway. Via this pathway telomerase-negative cancer cells can maintain their telomeres and with it their unlimited proliferative potential. A hallmark of ALT cells is the colocalization of promyelocytic leukemia (PML) nuclear bodies with telomeres to form ALT-associated PML nuclear bodies (APBs). In our screen, the presence of APBs was used as a marker to identify proteins required for the ALT mechanism. A cell-based assay and an automatic confocal image acquisition procedure were established. Using automatic image analysis based on 3D parametric intensity models to identify APBs, we conducted an unbiased and quantitative analysis of nine different candidate genes. A comparison with the literature and manual analysis of the gene knockdown demonstrates the reliability of our approach. It extends the available repertoire of high-content screening to studies of cellular colocalizations and allows the identification of candidate genes for the ALT mechanism that represent possible targets for cancer therapy.

PMID:
21648092
DOI:
10.1002/biot.201000474
[Indexed for MEDLINE]

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