A powerful method combining homologous recombination and site-specific recombination for targeted mutagenesis in Drosophila
A powerful method combining homologous recombination and site-specific recombination for targeted mutagenesis in Drosophila
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DOI：
10.1073/pnas.0805843105
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发表时间：
2008-09-16
影响因子：
11.1
通讯作者：
Rong, Yikang S.
中科院分区：
文献类型：
--
作者：
Gao, Guanjun;McMahon, Conor;Rong, Yikang S.
基因打靶为剖析基因功能提供了一个强有力的工具。然而，重复靶向单一基因座仍然是一种实践，主要限于单细胞生物，提供简单的靶向方法。我们开发了一种有效的方法来重复靶向果蝇中的单个位点。在我们称之为“位点特异性整合酶介导的重复靶向”（SIRT）的该方法中，首先通过同源重组将噬菌体phiC31整合酶的attP附着位点靶向至感兴趣的基因附近。该基因的所有后续修饰通过phiC31介导的携带attB附着位点和所需突变的质粒整合来引入。这种高效整合导致靶基因座的串联复制，然后可能通过有效的“单链退火”机制，用DNA双链断裂（DSB）诱导，将其还原成携带突变的单拷贝。我们使用SIRT在果蝇nbs基因中产生了一系列的六个突变，从单个氨基酸替换和小的框内缺失到基因的完全缺失。由于SIRT的所有组成部分在许多不同的生物体中都有功能，因此它很容易适应其他多细胞生物。
Gene targeting provides a powerful tool for dissecting gene function. However, repeated targeting of a single locus remains a practice mostly limited to unicellular organisms that afford simple targeting methodologies. We developed an efficient method to repeatedly target a single locus in Drosophila. In this method, which we term "site-specific integrase mediated repeated targeting" (SIRT), an attP attachment site for the phage phiC31 integrase is first targeted to the vicinity of the gene of interest by homologous recombination. All subsequent modifications of that gene are introduced by phiC31-mediated integration of plasmids carrying an attB attachment site and the desired mutation. This highly efficient integration results in a tandem duplication of the target locus, which is then reduced into a single copy carrying the mutation, likely by the efficient "single strand annealing" mechanism, induced with a DNA double-strand break (DSB). We used SIRT to generate a series of six mutations in the Drosophila nbs gene, ranging from single amino acid replacements and small in-frame deletions to complete deletion of the gene. Because all of the components of SIRT are functional in many different organisms, it is readily adaptable to other multicellular organisms.