Next-generation site-directed transgenesis in the malaria vector mosquito Anopheles gambiae: self-docking strains expressing germline-specific phiC31 integrase.
Next-generation site-directed transgenesis in the malaria vector mosquito Anopheles gambiae: self-docking strains expressing germline-specific phiC31 integrase.
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DOI：
10.1371/journal.pone.0059264
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Eggleston P
中科院分区：
文献类型：
--
作者：
Meredith JM;Underhill A;McArthur CC;Eggleston P
蚊子传播的疾病对全球健康具有破坏性影响，由于现有控制措施的困难和气候变化的影响，情况变得复杂。对疾病传播具有抗性的转基因蚊子被认为在提供新的控制策略方面具有巨大潜力。链霉菌噬菌体phiC31整合酶系统已经成功地适应了一系列昆虫的定点转基因整合，从而克服了由于大小限制和转座子介导转化相关的随机整合所带来的许多限制。利用这项技术，我们之前发表了第一篇针对冈比亚按蚊（人类疟疾的主要媒介）的定点转化。蚊子最初被改造成在一个确定的基因组位置加入phiC31对接位点。基因修饰的第二阶段随后实现了抗疟疾效应基因的定点整合。在当前的出版物中，我们报告了在冈比亚按蚊自对接菌株产生后，提高了phiC31整合酶系统的效率和效用。在冈比亚按蚊纳米基因调控区域的控制下，四个独立的菌株在三条不同染色体臂上已知的对接位点表达整合酶。由此产生的蛋白质在卵母细胞后部积累，在种系发育部位提供整合酶活性。两种整合酶表达水平显著不同的自对接菌株进行了位点定向转基因整合评估，发现它们的存活率和转化效率大大提高。在与疟疾的斗争中，必须建立广泛的抗疟疾效应基因和组织特异性启动子库，以调节它们的表达，从而确定那些以最小适应度成本提供最大效果的基因。我们在这里描述的改进技术将促进效应转基因的比较研究，允许做出可能导致传播阻断的知情选择。
Diseases transmitted by mosquitoes have a devastating impact on global health and the situation is complicated due to difficulties with both existing control measures and the impact of climate change. Genetically modified mosquitoes that are refractory to disease transmission are seen as having great potential in the delivery of novel control strategies. The Streptomyces phage phiC31 integrase system has been successfully adapted for site-directed transgene integration in a range of insects, thus overcoming many limitations due to size constraints and random integration associated with transposon-mediated transformation. Using this technology, we previously published the first site-directed transformation of Anopheles gambiae, the principal vector of human malaria. Mosquitoes were initially engineered to incorporate the phiC31 docking site at a defined genomic location. A second phase of genetic modification then achieved site-directed integration of an anti-malarial effector gene. In the current publication we report improved efficiency and utility of the phiC31 integrase system following the generation of Anopheles gambiae self-docking strains. Four independent strains, with docking sites at known locations on three different chromosome arms, were engineered to express integrase under control of the regulatory regions of the nanos gene from Anopheles gambiae. The resulting protein accumulates in the posterior oocyte to provide integrase activity at the site of germline development. Two self-docking strains, exhibiting significantly different levels of integrase expression, were assessed for site-directed transgene integration and found to demonstrate greatly improved survival and efficiency of transformation. In the fight against malaria, it is imperative to establish a broad repertoire of both anti-malarial effector genes and tissue-specific promoters to regulate their expression, enabling those offering maximum effect with minimum fitness cost to be identified. The improved technology we describe here will facilitate comparative studies of effector transgenes, allowing informed choices to be made that potentially lead to transmission blockade.
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