Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes
Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes
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DOI：
10.1038/ng.101
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发表时间：
2008-04-01
期刊：
影响因子：
30.8
通讯作者：
Perrimon, Norbert
中科院分区：
文献类型：
--
作者：
Markstein, Michele;Pitsouli, Chrysoula;Perrimon, Norbert
创造精确表达的转基因的主要障碍在于围绕它们的宿主染色质的表观遗传效应。在这里，我们提出了一个策略来克服这个问题，采用Gal4诱导荧光素酶测定系统地量化宿主染色质的位置效应和绝缘子的能力，以抵消这些影响在phiC31整合位点随机分布在整个果蝇基因组。我们确定的基因座，可以利用提供精确剂量的转基因表达的特定组织。此外，我们发现了吉普赛逆转录病毒绝缘子的一个以前未被认识到的属性，以提高基因表达的水平比大多数或可能所有的非绝缘位点，在每一个组织测试。这些发现提供了第一个机会，以创建一个电池的转基因，可以可靠地在几乎任何组织中的高水平，通过整合在一个单一的基因座，相反，工程控制的表型等位基因系列，通过利用几个基因座。我们的方法的通用性使其适用于其他模型系统，以确定和修改基因座的最佳转基因表达。
A major obstacle to creating precisely expressed transgenes lies in the epigenetic effects of the host chromatin that surrounds them. Here we present a strategy to overcome this problem, employing a Gal4-inducible luciferase assay to systematically quantify position effects of host chromatin and the ability of insulators to counteract these effects at phiC31 integration loci randomly distributed throughout the Drosophila genome. We identify loci that can be exploited to deliver precise doses of transgene expression to specific tissues. Moreover, we uncover a previously unrecognized property of the gypsy retrovirus insulator to boost gene expression to levels severalfold greater than at most or possibly all un-insulated loci, in every tissue tested. These findings provide the first opportunity to create a battery of transgenes that can be reliably expressed at high levels in virtually any tissue by integration at a single locus, and conversely, to engineer a controlled phenotypic allelic series by exploiting several loci. The generality of our approach makes it adaptable to other model systems to identify and modify loci for optimal transgene expression.