Untangling the relationships between DNA repair pathways by silencing more than 20 DNA repair genes in human stable clones
Untangling the relationships between DNA repair pathways by silencing more than 20 DNA repair genes in human stable clones
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DOI：
10.1093/nar/gkm195
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发表时间：
2007-06-01
影响因子：
14.9
通讯作者：
Biard, D. S. F.
中科院分区：
文献类型：
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作者：
Biard, D. S. F.
长期以来，人们一直致力于阐明细胞对遗传毒性损伤的协调反应。鉴于很难获得同源的人类生物样品，我已经建立了一套稳定的人类克隆，其中一个DNA修复基因已经通过RNA干扰稳定沉默。我使用了pEBVsiRNA质粒，它极大地增强了人类细胞中的长期基因沉默。我的老克隆体在培养中达到了4500天。敲低的HeLa克隆在培养中长时间保持基因沉默表型，表明我能够模拟来自癌症易感综合征的细胞。我已经沉默了超过420个基因作为传感器/转换器（ATM，ATR，Rad 50，NBS 1，MRE 11，PARG和KIN 17），或不同的DNA修复途径。在HeLa细胞中，我已经关闭了参与核苷酸切除修复（XPA，XPC，hHR 23 A，hHR 23 B，CSA和CSB），非同源末端连接（DNA-PKcs，XRCC 4和Ligase IV），同源重组修复（Rad 51和Rad 54）或碱基切除修复（Ogg 1和Ligase III）的基因的表达。这些细胞显示出预期的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，没有使用病毒载体，以及随之而来的伦理和安全问题。
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 4500 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 > 420 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.