Site-specific transformation of Drosophila via φC31 integrase-mediated cassette exchange
Site-specific transformation of Drosophila via φC31 integrase-mediated cassette exchange
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DOI：
10.1534/genetics.106.056945
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发表时间：
2006-06-01
期刊：
影响因子：
3.3
通讯作者：
Wu, C. -ting
中科院分区：
文献类型：
--
作者：
Bateman, Jack R.;Lee, Anne M.;Wu, C. -ting
位置效应会使转基因分析变得复杂。当比较随机插入不同基因组位置并因此受到不同位置效应影响的转基因时，尤其如此。在此，我们介绍一种利用φC31整合酶系统结合重组酶介导的盒式交换（RMCE）将转基因构建体精确靶向果蝇预定基因组位点的方法。我们使用两个供体盒证明了该系统的可行性，一个携带黄色基因，另一个携带绿色荧光蛋白基因。在所有测试的四个基因组位点上，我们观察到供体盒与携带迷你白眼基因的整合靶盒发生了交换。此外，由于RMCE介导的供体盒整合必然伴随着靶盒的丢失，我们能够通过迷你白眼颜色的缺失来识别整合体。重要的是，该技术的这一特性将允许无标记构建体整合到果蝇中，即使是那些缺乏功能基因的构建体。因此，φC31整合酶介导的RMCE应该会极大地促进转基因分析，并允许新的实验设计。
Position effects can complicate transgene analyses. This is especially true when comparing transgenes that have inserted randomly into different genomic positions and are therefore subject to varying position effects. Here, we introduce a method for the precise targeting of transgenic constructs to predetermined genomic sites in Drosophila using the phi C31 integrase system in conjunction with recombinase-mediated cassette exchange (RMCE). We demonstrate the feasibility of this system using two donor cassettes, one carrying the Yellow gene and the other carrying GFP At all four genomic sites tested, we observed exchange of donor cassettes with an integrated target cassette carrying the mini-white gene. Furthermore, because RMCE-mediated integration of the donor cassette is necessarily accompanied by loss of the target cassette, we were able to identify integrants simply by the loss of mini-white eye color. Importantly, this feature of the technology will permit integration of unmarked constructs into Drosophila, even those lacking functional,genes. Thus, phi C31 integrase-mediated RMCE should greatly facilitate transgene analysis as well as permit new experimental designs.