Pradhan Barun, Kauppi Liisa
Systems Oncology Research Program, University of Helsinki; Department of Biochemistry and Developmental Biology, Medicum, University of Helsinki.
Systems Oncology Research Program, University of Helsinki; Department of Biochemistry and Developmental Biology, Medicum, University of Helsinki;
J Vis Exp. 2019 Jul 27(149). doi: 10.3791/59880.
Long interspersed nuclear elements 1 (LINE-1s) are the only family of mobile genetic elements in the human genome that can move autonomously. They do so by a process called retrotransposition wherein they transcribe to form an mRNA intermediate which is then consequently inserted into the genome by reverse transcription. Despite being silent in normal cells, LINE-1s are highly active in different epithelial tumors. De novo LINE-1 insertions can potentially drive tumorigenesis, and hence it is important to systematically study LINE-1 retrotransposition in cancer. Out of ~150 retrotransposition-competent LINE-1s present in the human genome, only a handful of LINE-1 loci, also referred to as "hot" LINE-1s, account for the majority of de novo LINE-1 insertion in different cancer types. We have developed a simple polymerase chain reaction (PCR)-based method to monitor retrotransposition activity of these hot LINE-1s. This method, based on long-distance inverse (LDI)-PCR, takes advantage of 3´ transduction, a mechanism by which a LINE-1 mobilizes its flanking non-repetitive region, which can subsequently be used to identify de novo LINE-1 3´ transduction events stemming from a particular hot LINE-1.
长散在核元件1（LINE-1）是人类基因组中唯一能够自主移动的可移动遗传元件家族。它们通过一种称为逆转座的过程来实现移动，在此过程中，它们转录形成mRNA中间体，然后通过逆转录被插入到基因组中。尽管LINE-1在正常细胞中是沉默的，但它们在不同的上皮肿瘤中高度活跃。从头LINE-1插入可能会驱动肿瘤发生，因此系统地研究癌症中的LINE-1逆转座很重要。在人类基因组中存在的约150个具有逆转座能力的LINE-1中，只有少数LINE-1位点，也被称为“热点”LINE-1，占不同癌症类型中从头LINE-1插入的大部分。我们开发了一种基于简单聚合酶链反应（PCR）的方法来监测这些热点LINE-1的逆转座活性。这种基于长距离反向（LDI）-PCR的方法利用了3´转导，这是一种LINE-1移动其侧翼非重复区域的机制，随后可用于识别源自特定热点LINE-1的从头LINE-1 3´转导事件。
