Nucleic Acids Research Advance Access originally published online on May 5, 2007
Nucleic Acids Research 2007 35(11):3535-3550; doi:10.1093/nar/gkm195
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Nucleic Acids Research, 2007, Vol. 35, No. 11 3535-3550
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
Untangling the relationships between DNA repair pathways by silencing more than 20 DNA repair genes in human stable clones
Laboratoire de Génétique de la Radiosensibilité, Institut de Radiobiologie Cellulaire et Moléculaire, Direction des Sciences du Vivant, Commissariat à l’Energie Atomique (CEA), BP 6, Fontenay-aux-Roses 92265, France
*To whom correspondence should be addressed. Email: denis.biard{at}cea.fr
Received January 25, 2007. Revised March 20, 2007. Accepted March 20, 2007.
Much effort has long been devoted to unraveling the coordinated cellular response to genotoxic insults. In view of the difficulty of obtaining human biological samples of homogeneous origin, I have established a set of stable human clones where one DNA repair gene has been stably silenced by means of RNA interference. I used pEBVsiRNA plasmids that greatly enhance long-term gene silencing in human cells. My older clones reached >500 days in culture. Knock-down HeLa clones maintained a gene silencing phenotype for an extended period in culture, demonstrating that I was able to mimic cells from cancer-prone syndromes. I have silenced >20 genes acting as sensors/transducers (ATM, ATR, Rad50, NBS1, MRE11, PARG and KIN17), or of different DNA repair pathways. In HeLa cells, I have switched off the expression of genes involved in nucleotide excision repair (XPA, XPC, hHR23A, hHR23B, CSA and CSB), nonhomologous end-joining (DNA-PKcs, XRCC4 and Ligase IV), homologous recombination repair (Rad51 and Rad54), or base excision repair (Ogg1 and Ligase III). These cells displayed the expected DNA repair phenotype. We could envisage untangling the complex network between the different DNA repair pathways. In this study, no viral vehicles, with their attendant ethical and safety concerns, were used.
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