A novel target-specific gene delivery system combining baculovirus and sequence-specific long interspersed nuclear elements
A novel target-specific gene delivery system combining baculovirus and sequence-specific long interspersed nuclear elements
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DOI：
10.1016/j.virusres.2007.03.014
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发表时间：
2007-07-01
期刊：
影响因子：
5
通讯作者：
Fujiwara, Haruhiko
中科院分区：
文献类型：
--
作者：
Kawashima, Tomoko;Osanai, Mizuko;Fujiwara, Haruhiko
转座因子在体细胞和胚系转化中具有重要的应用价值。然而，由于对转座机制的信息不多，长散布核元件（LINES）尚未被使用。我们已经开发了一种新的基因传递系统相结合的杆状病毒AcNPV和两个家蚕LINES，SART 1和R1，这两个LINES分别整合到端粒重复序列和28S核糖体DNA的特定序列。将重组到AcNPV中的增强型绿色荧光蛋白基因导入家蚕5龄幼虫中，利用PCR扩增和测序技术，在感染后72 h观察到LINEs的靶特异性反转录转座。端粒和28S rDNA特异性转位发生在所有九个组织测试，包括卵巢和睾丸。这是第一次在活体幼虫中展示位点特异性基因传递。插入效率取决于注射用病毒滴度和家蚕宿主株。利用该系统，我们成功地检测到了逆转置序列的代际传播。此外，AcNPV介导的SART 1也转座到另一种鳞翅目昆虫Orgyia recens的端粒中，这表明该系统可用于多种AcNPV感染性昆虫。通过病毒介导的LINE的位点特异性基因递送将是一种潜在的基因治疗工具，以避免有害的意外插入。(C)2007 Elsevier B.V.保留所有权利。
Transposable elements are valuable for somatic and germ-line transformation. However, long interspersed nuclear elements (LINEs) have not been used because of poor information on the transposition mechanism. We have developed a novel gene delivery system combining baculovirus AcNPV and two silkworm LINEs, SART1 and R1, which integrate into specific sequences of telomeric repeats and 28S ribosomal DNA, respectively. When two LINEs containing the enhanced green fluorescent protein gene recombined into AcNPV were infected into fifth instar larvae of the silkworm, we observed target-specific retrotransposition of LINEs at 72 h post-infection, using polymerase chain reaction amplification and sequencing. Telomere- and 28S rDNA-specific transposition occurred in all nine tissues tested, including the ovary and testis. This is the first demonstration of site-specific gene delivery in living larvae. Insertion efficiencies were dependent on the virus titer for injection and the host strains of Bombyx mori. Using this system, we successfully detected the intergeneration transmission of retrotransposed sequences. In addition, AcNPV-mediated SART1 also transposed into telomere of another lepidopteran, Orgyia recens, suggesting that this system is useful for a wide variety of AcNPV-infectious insects. Site-specific gene delivery by virus-mediated LINE will be a potential gene therapy tool to avoid harmful unexpected insertions. (C) 2007 Elsevier B.V. All rights reserved.