Untangling the relationships between DNA repair pathways by silencing more than 20 DNA repair genes in human stable clones.
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.
Biard
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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. denis.biard@cea.fr
D017766:Acid Anhydride Hydrolases; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D002999:Clone Cells; D004249:DNA Damage; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000037:antagonists & inhibitors / Q000235:genetics; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D005047:Etoposide / Q000633:toxicity; D005720:Gamma Rays; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D000076228:MRE11 Homologue Protein; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D034622:RNA Interference; D014466:Ultraviolet Rays
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; C121766:MRE11 protein, human; C112156:NBN protein, human; D009687:Nuclear Proteins; D005047:Etoposide; D000076228:MRE11 Homologue Protein; D017766:Acid Anhydride Hydrolases; C112158:RAD50 protein, human; D045643:DNA Repair Enzymes