Honda Hiroshi, Ichiyanagi Kenji, Suzuki Jun, Ono Takao, Koyama Hideki, Kajikawa Masaki, Okada Norihiro
Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259-B-21 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8501, Japan.
Gene. 2007 Jun 15;395(1-2):116-24. doi: 10.1016/j.gene.2007.02.017. Epub 2007 Mar 14.
Long interspersed elements (LINEs) are autonomous transposable elements that proliferate via retrotransposition, which involves reverse transcription of LINE RNAs. It is anticipated that LINE retrotransposition requires both LINE-encoded proteins and host-encoded proteins. However, identification of the host factors, their roles, and the steps at which they act on retrotransposition are poorly understood because of the lack of an appropriate genetic system to study LINE retrotransposition in a series of mutant hosts. To construct such a genetic system, we applied the retrotransposition-indicative cassette method to DT40 cells, a chicken cell line for which a variety of isogenic mutants have been established by gene targeting. Because DT40 cells are non-adherent, we utilized a selective soft agarose medium to allow the formation of colonies of cells that had undergone LINE retrotransposition. Colony formation was completely dependent on the activities of the LINE-encoded proteins and on the presence of the essential 3' region of the LINE RNA. Moreover, the selected colonies indeed carried retrotransposed LINE copies in their chromosomes, with integration features similar to those of genomic (native) LINE copies. This method thus allows the authentic selection of LINE-retrotransposed cells and the approximate recapitulation of retrotransposition events that occur in nature. Therefore, the DT40 cell system established here provides a powerful tool for the elucidation of LINE retrotransposition pathways, the host factors involved, and their roles.
长散在元件（LINEs）是通过逆转座进行增殖的自主转座元件，逆转座过程涉及LINE RNA的逆转录。据推测，LINE逆转座需要LINE编码的蛋白质和宿主编码的蛋白质。然而，由于缺乏合适的遗传系统来在一系列突变宿主中研究LINE逆转座，目前对于宿主因子的鉴定、它们的作用以及它们作用于逆转座的步骤了解甚少。为了构建这样一个遗传系统，我们将逆转座指示盒法应用于DT40细胞，这是一种鸡细胞系，通过基因打靶已建立了多种同基因突变体。由于DT40细胞不贴壁，我们利用选择性软琼脂培养基来形成经历了LINE逆转座的细胞集落。集落形成完全依赖于LINE编码蛋白质的活性以及LINE RNA必需的3'区域的存在。此外，所选集落在其染色体中确实携带了逆转座的LINE拷贝，其整合特征与基因组（天然）LINE拷贝相似。因此，这种方法能够真正地筛选出LINE逆转座的细胞，并大致重现自然发生的逆转座事件。所以，这里建立的DT40细胞系统为阐明LINE逆转座途径、所涉及的宿主因子及其作用提供了一个强大的工具。
