Biard D S F
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.
Nucleic Acids Res. 2007;35(11):3535-50. doi: 10.1093/nar/gkm195. Epub 2007 May 5.
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.
长期以来，人们付出了巨大努力来揭示细胞对基因毒性损伤的协同反应。鉴于获取同源人类生物样本的困难，我建立了一组稳定的人类克隆细胞系，其中一个DNA修复基因已通过RNA干扰被稳定沉默。我使用了pEBVsiRNA质粒，它能极大地增强人类细胞中的长期基因沉默效果。我的早期克隆细胞在培养中存活了超过500天。敲低的HeLa克隆细胞在培养中长时间保持基因沉默表型，这表明我能够模拟易患癌症综合征患者的细胞。我已经沉默了20多个作为传感器/转导器（ATM、ATR、Rad50、NBS1、MRE11、PARG和KIN17）或参与不同DNA修复途径的基因。在HeLa细胞中，我关闭了参与核苷酸切除修复（XPA、XPC、hHR23A、hHR23B、CSA和CSB）、非同源末端连接（DNA-PKcs、XRCC4和连接酶IV）、同源重组修复（Rad51和Rad54）或碱基切除修复（Ogg1和连接酶III）的基因的表达。这些细胞表现出预期的DNA修复表型。我们可以设想解开不同DNA修复途径之间的复杂网络。在这项研究中，没有使用带有相关伦理和安全问题的病毒载体。
