Long-distance effects of insertional mutagenesis.
Long-distance effects of insertional mutagenesis.
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DOI：
10.1371/journal.pone.0015832
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发表时间：
2011-01-05
期刊：
影响因子：
3.7
通讯作者：
Kandel ES
中科院分区：
文献类型：
--
作者：
Singhal R;Deng X;Chenchik AA;Kandel ES
最常见的哺乳动物细胞基因工程系统与修饰细胞的插入诱变有关。插入诱变也是一种为基因发现项目产生随机改变的流行方法。更好地理解插入诱变剂内结构元件的相互作用以及这些元件影响距插入位点不同距离的宿主基因的能力具有相当大的实际重要性。我们观察到，在慢病毒构建体中，在内部 CMV 启动子/增强子区域启动并包含剪接供体位点的转录本能够延伸经过共线病毒 LTR 并捕获宿主基因的外显子，而自然存在于 LTR 中的多腺苷酸化信号则被剪接掉。出乎意料的是，当利用这种现象的载体产生NF-κB活性升高的突变体时，我们发现了突变体，其表型归因于插入对距离插入位点数十甚至数百千碱基的基因的影响。这种效应不是由 CMV 驱动的转录物引起的，而是对插入片段的功能抑制敏感。有趣的是，尽管存在长距离效应，但最接近插入片段的基因座的表达似乎不受影响。我们得出的结论是，当逆转录病毒LTR中的多腺苷酸化信号在内含子内发生时，对于杂合转录物的形成来说是一个低效的屏障，并且含有强增强子的载体可能选择性地影响远离其插入位点的基因的功能。在进行实验或治疗性转导时必须考虑这些现象。特别是，插入诱变的远距离效应使疾病相关逆转录病毒整合靶点列表的相关性受到质疑，这些靶点未经过功能验证。
Most common systems of genetic engineering of mammalian cells are associated with insertional mutagenesis of the modified cells. Insertional mutagenesis is also a popular approach to generate random alterations for gene discovery projects. A better understanding of the interaction of the structural elements within an insertional mutagen and the ability of such elements to influence host genes at various distances away from the insertion site is a matter of considerable practical importance. We observed that, in the context of a lentiviral construct, a transcript, which is initiated at an internal CMV promoter/enhancer region and incorporates a splice donor site, is able to extend past a collinear viral LTR and trap exons of host genes, while the polyadenylation signal, which is naturally present in the LTR, is spliced out. Unexpectedly, when a vector, which utilizes this phenomenon, was used to produce mutants with elevated activity of NF-κB, we found mutants, which owed their phenotype to the effect of the insert on a gene located tens or even hundreds of kilobases away from the insertion site. This effect did not result from a CMV-driven transcript, but was sensitive to functional suppression of the insert. Interestingly, despite the long-distance effect, expression of loci most closely positioned to the insert appeared unaffected. We concluded that a polyadenylation signal in a retroviral LTR, when occurring within an intron, is an inefficient barrier against the formation of a hybrid transcript, and that a vector containing a strong enhancer may selectively affect the function of genes far away from its insertion site. These phenomena have to be considered when experimental or therapeutic transduction is performed. In particular, the long-distance effects of insertional mutagenesis bring into question the relevance of the lists of disease-associated retroviral integration targets, which did not undergo functional validation.
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DOI：
10.1073/pnas.160265197
发表时间：
2000-09-12
影响因子：
11.1
作者：
Li, XX;Commane, M;Stark, GR
通讯作者：
Stark, GR
影响因子：
5.3
作者：
DUBRIDGE, RB;TANG, P;CALOS, MP
通讯作者：
CALOS, MP
DOI：
10.1073/pnas.0306812101
发表时间：
2004-01-06
影响因子：
11.1
作者：
Sathe, SS;Sizemore, N;Stark, GR
通讯作者：
Stark, GR
影响因子：
56.9
作者：
SWAIN, A;COFFIN, JM
通讯作者：
COFFIN, JM
影响因子：
20.3
作者：
Zhang F;Thornhill SI;Howe SJ;Ulaganathan M;Schambach A;Sinclair J;Kinnon C;Gaspar HB;Antoniou M;Thrasher AJ
通讯作者：
Thrasher AJ