A new system for analyzing LINE retrotransposition in the chicken DT40 cell line widely used for reverse genetics
A new system for analyzing LINE retrotransposition in the chicken DT40 cell line widely used for reverse genetics
复制标题
DOI：
10.1016/j.gene.2007.02.017
复制
发表时间：
2007-06-15
期刊：
影响因子：
3.5
通讯作者：
Okada, Norihiro
中科院分区：
文献类型：
--
作者：
Honda, Hiroshi;Ichiyanagi, Kenji;Okada, Norihiro
长散布元件（LINE）是通过反转录转座增殖的自主转座元件，其涉及LINERNA的逆转录。预计LINE逆转录转座需要LINE编码的蛋白质和宿主编码的蛋白质。然而，鉴定宿主因子，它们的作用，以及它们作用于反转录转座的步骤知之甚少，因为缺乏适当的遗传系统来研究LINE在一系列突变宿主中的反转录转座。为了构建这样一个遗传系统，我们应用反转录转座指示盒的方法，以DT 40细胞，鸡细胞系的各种同基因突变体已建立基因打靶。因为DT 40细胞是非粘附性的，我们利用选择性软琼脂培养基来允许已经经历LINE逆转录转座的细胞的集落形成。菌落形成完全依赖于LINE编码蛋白的活性和LINE RNA的必需3'区的存在。此外，选择的菌落确实在其染色体中携带逆转录转座的LINE拷贝，其整合特征类似于基因组（天然）LINE拷贝的整合特征。因此，该方法允许真实地选择LINE逆转录转座的细胞和自然界中发生的逆转录转座事件的近似重演。因此，DT 40细胞系统建立这里提供了一个强大的工具，阐明LINE反转录转座途径，涉及的宿主因子，以及它们的作用。(c)2007 Elsevier B.V.保留所有权利。
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 forination 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. (c) 2007 Elsevier B.V. All rights reserved.