Short-term cytotoxic effects and long-term instability of RNAi delivered using lentiviral vectors.
Short-term cytotoxic effects and long-term instability of RNAi delivered using lentiviral vectors.
复制标题
DOI：
10.1186/1471-2199-5-9
复制
发表时间：
2004-08-03
影响因子：
--
通讯作者：
Kruithof EK
中科院分区：
文献类型：
--
作者：
Fish RJ;Kruithof EK
RNA干扰（RNAi）可以有效地降低哺乳动物细胞中靶基因的表达，并被广泛用于功能丧失的研究。最近的一些报道表明，用于介导RNAi的短双链rna （dsRNAs）也可以诱导基于干扰素的应答，导致许多干扰素应答基因的表达改变。脱靶基因沉默也被描述过，这对某些基于rnai的基因功能研究方法的有效性提出了质疑。我们使用慢病毒载体靶向纤溶酶原激活物抑制剂-2 (PAI-2或SERPINB2) mRNA，以传递U6启动子驱动的PAI-2靶向短发夹RNA （shRNA）表达。据报道，PAI-2具有抗凋亡活性，因此内源性表达的减少可能会使细胞对程序性细胞死亡更敏感。正如预期的那样，当我们用载体衍生的shRNA靶向PAI-2 mRNA时，我们遇到了细胞毒性表型。然而，这种预测的表型是shRNA表达的一种强有力的非特异性效应，因为靶蛋白的功能性过表达未能挽救表型。通过减少shRNA长度或修改其序列，我们维持了PAI-2的沉默，并降低了细胞毒性，但没有消除细胞毒性。21个互补核苷酸（21粒）或更多的ShRNA增加了低聚腺苷酸合成酶-1 （OAS1）干扰素应答基因的表达。19 mer shRNA对OAS1表达无影响，但对细胞生长有长期的选择性压力。通过降低慢病毒载体滴度，我们能够降低shRNA的表达和OAS1的诱导，而对基因沉默的效果没有重大影响。我们的数据证明了shRNAs在人类肿瘤细胞系中表达的快速细胞毒性作用。似乎有21个互补核苷酸的临界值，低于这个临界值时，靶基因保持沉默，不会产生干扰素应答。通过改变shRNA序列或载体滴度可以降低细胞毒性或OAS1诱导，但在延长的细胞培养中，尽管rnai诱导的转基因盒中持续表达标记基因，但无法维持稳定的基因沉默。这些结果强调了在哺乳动物细胞系统中立即和长期使用RNAi的必要性。
RNA interference (RNAi) can potently reduce target gene expression in mammalian cells and is in wide use for loss-of-function studies. Several recent reports have demonstrated that short double-stranded RNAs (dsRNAs), used to mediate RNAi, can also induce an interferon-based response resulting in changes in the expression of many interferon-responsive genes. Off-target gene silencing has also been described, bringing into question the validity of certain RNAi-based approaches for studying gene function. We have targeted the plasminogen activator inhibitor-2 (PAI-2 or SERPINB2) mRNA using lentiviral vectors for delivery of U6 promoter-driven PAI-2-targeted short hairpin RNA (shRNA) expression. PAI-2 is reported to have anti-apoptotic activity, thus reduction of endogenous expression may be expected to make cells more sensitive to programmed cell death. As expected, we encountered a cytotoxic phenotype when targeting the PAI-2 mRNA with vector-derived shRNA. However, this predicted phenotype was a potent non-specific effect of shRNA expression, as functional overexpression of the target protein failed to rescue the phenotype. By decreasing the shRNA length or modifying its sequence we maintained PAI-2 silencing and reduced, but did not eliminate, cytotoxicity. ShRNA of 21 complementary nucleotides (21 mers) or more increased expression of the oligoadenylate synthase-1 (OAS1) interferon-responsive gene. 19 mer shRNA had no effect on OAS1 expression but long-term selective pressure on cell growth was observed. By lowering lentiviral vector titre we were able to reduce both expression of shRNA and induction of OAS1, without a major impact on the efficacy of gene silencing. Our data demonstrate a rapid cytotoxic effect of shRNAs expressed in human tumor cell lines. There appears to be a cut-off of 21 complementary nucleotides below which there is no interferon response while target gene silencing is maintained. Cytotoxicity or OAS1 induction could be reduced by changing shRNA sequence or vector titre, but stable gene silencing could not be maintained in extended cell culture despite persistent marker gene expression from the RNAi-inducing transgene cassette. These results underscore the necessity of careful controls for immediate and long-term RNAi use in mammalian cell systems.
登录
查看更多内容
影响因子：
46.9
作者：
Jackson, AL;Bartz, SR;Linsley, PS
通讯作者：
Linsley, PS
DOI：
10.1073/pnas.1630797100
发表时间：
2003-08-19
影响因子：
11.1
作者：
Zeng, Y;Yi, R;Cullen, BR
通讯作者：
Cullen, BR
影响因子：
4.8
作者：
DICKINSON, JL;BATES, EJ;ANTALIS, TM
通讯作者：
ANTALIS, TM
DOI：
10.1073/pnas.232688199
发表时间：
2003-01-07
影响因子：
11.1
作者：
Qin, XF;An, DS;Baltimore, D
通讯作者：
Baltimore, D
影响因子：
30.8
作者：
Bridge, AJ;Pebernard, S;Iggo, R
通讯作者：
Iggo, R