Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, New York, United States of America.
PLoS One. 2011 Jan 5;6(1):e15832. doi: 10.1371/journal.pone.0015832.
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.
METHODOLOGY/PRINCIPAL FINDINGS: 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.
CONCLUSIONS/SIGNIFICANCE: 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.
大多数哺乳动物细胞的基因工程系统都与修饰细胞的插入突变有关。插入突变也是用于基因发现项目的随机改变的常用方法。更好地了解插入突变体的结构元件之间的相互作用，以及这些元件在插入位点的各种距离处影响宿主基因的能力，是一个非常重要的实际问题。
方法/主要发现：我们观察到，在慢病毒构建体的情况下，从内部 CMV 启动子/增强子区域起始并包含剪接供体位点的转录本能够延伸到共线性病毒 LTR 之外，并捕获宿主基因的外显子，而聚腺苷酸化信号，天然存在于 LTR 中，被剪接出来。出乎意料的是，当使用利用这种现象的载体来产生 NF-κB 活性升高的突变体时，我们发现突变体的表型归因于插入对位于插入位点数十甚至数百千碱基之外的基因的影响。这种效应不是由 CMV 驱动的转录物引起的，而是对插入的功能抑制敏感。有趣的是，尽管存在远距离效应，但与插入最接近的基因座的表达似乎不受影响。
结论/意义：我们得出结论，当逆转录病毒 LTR 中的聚腺苷酸化信号位于内含子时，它是形成杂交转录本的低效屏障，并且包含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，插入突变的远距离效应使得疾病相关逆转录病毒整合靶目标的列表的相关性受到质疑，这些靶目标没有经过功能验证。
