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A three‐dimensional colocalization RNA interference screening platform to elucidate the alternative lengthening of telomeres pathway
Author(s) -
Osterwald Sarah,
Wörz Stefan,
Reymann Jürgen,
Sieckmann Frank,
Rohr Karl,
Erfle Holger,
Rippe Karsten
Publication year - 2012
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201000474
Subject(s) - colocalization , telomere , telomerase , biology , confocal microscopy , gene knockdown , microbiology and biotechnology , rna interference , computational biology , confocal , gene , rna , genetics , physics , optics
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.