Knapp Jon-Michael, Chung Phuong, Simpson Julie H
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147.
Genetics. 2015 Apr;199(4):919-34. doi: 10.1534/genetics.114.173187. Epub 2015 Feb 12.
Transgenesis in numerous eukaryotes has been facilitated by the use of site-specific integrases to stably insert transgenes at predefined genomic positions (landing sites). However, the utility of integrase-mediated transgenesis in any system is constrained by the limited number and variable expression properties of available landing sites. By exploiting the nonstandard recombination activity exhibited by a phiC31 integrase mutant, we developed a rapid and inexpensive method for isolating landing sites that exhibit desired expression properties. Additionally, we devised a simple technique for constructing arrays of transgenes at a single landing site, thereby extending the utility of previously characterized landing sites. Using the fruit fly Drosophila melanogaster, we demonstrate the feasibility of these approaches by isolating new landing sites optimized to express transgenes in the nervous system and by building fluorescent reporter arrays at several landing sites. Because these strategies require the activity of only a single exogenous protein, we anticipate that they will be portable to species such as nonmodel organisms, in which genetic manipulation is more challenging, expediting the development of genetic resources in these systems.
在众多真核生物中，通过使用位点特异性整合酶将转基因稳定插入预定义的基因组位置（着陆位点），转基因技术得到了促进。然而，整合酶介导的转基因技术在任何系统中的应用都受到可用着陆位点数量有限和表达特性可变的限制。通过利用phiC31整合酶突变体表现出的非标准重组活性，我们开发了一种快速且廉价的方法来分离具有所需表达特性的着陆位点。此外，我们设计了一种简单的技术，用于在单个着陆位点构建转基因阵列，从而扩展了先前表征的着陆位点的用途。利用果蝇，我们通过分离优化用于在神经系统中表达转基因的新着陆位点以及在几个着陆位点构建荧光报告基因阵列，证明了这些方法的可行性。由于这些策略仅需要单一外源蛋白的活性，我们预计它们将适用于诸如非模式生物等物种，在这些物种中基因操作更具挑战性，从而加速这些系统中遗传资源的开发。
