Recombination-mediated cassette exchange in Aedes aegypti mosquitoes

描述（由申请人提供）：疟疾和登革出血热等病媒传播疾病仍然是巨大的公共卫生负担，仍然需要新的干预措施。基于遗传学的控制的新兴领域已经看到了许多最近的实验室成功，产生了抗病原体的蚊子菌株以及杀死雌性的菌株。转基因蚊子的发展仍然在很大程度上依赖于经典的转座因子转化，虽然最近的几份报告已经利用了位点特异性整合酶C31。虽然位点特异性允许研究者在相同的遗传环境中检查多个转基因，但attP-attB C31系统的不幸结果是，由于重组，整个细菌质粒整合到蚊子基因组中。这是不希望的，因为许多目前开发的转基因蚊子品系旨在作为特定的遗传干预以控制靶向的媒介传播的病原体，其中这种抗生素抗性基因可以转移到天然细菌物种。我们假设，重组酶介导的盒式交换（RMCE）可以是一种有效的手段，将转基因以位点特异性的方式传递到重要的疾病载体物种中，而不会产生共整合细菌序列的负面后果。因此，我们建议（1）测试一组异源特异性lox位点在登革热载体埃及伊蚊和疟疾载体冈比亚按蚊的胚胎中抵抗分子内重组的能力和促进RMCE的能力;（2）基于最佳候选异源特异性lox位点产生转基因对接株，并使用噬菌体P1 cre重组酶确定每个位点的RMCE率。保留进行位点特异性重组的能力，但避免细菌序列的整合并保持与基于TE的辅助质粒一样易于使用的这种替代系统可能会被更广泛地采用，并将有助于推动用于媒介传播疾病的新型基于遗传学的控制策略的领域。

DESCRIPTION (provided by applicant): Vector-borne diseases such as malaria and dengue hemorrhagic fever remain large public health burdens, and novel interventions are still needed. The emerging field of genetics-based control has seen a number of recent laboratory successes with the generation of pathogen- resistant mosquito strains as well as female-killing strains. The development of genetically modified mosquitoes still largely relies upon classical transposable element transformation, although several recent reports have made use of the site-specific integrase C31. While site- specificity allows the investigator to examine multiple transgenes in the same genetic environment, an unfortunate consequence of the attP-attB C31 system is that as a result of recombination the entire bacterial plasmid becomes integrated into the mosquito genome. This is undesirable, as many of the currently developed transgenic mosquito strains are intended as specific genetic interventions to control a targeted vector-borne pathogen where such antibiotic resistance genes could be transferred to native bacterial species. We hypothesize that recombinase-mediated cassette exchange (RMCE) can be an efficient means of delivering transgenes into important disease vector species in a site-specific manner, without the negative consequences of co-integrating bacterial sequences. As such, we propose to (1) test a panel of heterospecific lox sites for their ability to resist intramolecular recombination i cis and promote RMCE in the embryos of the dengue vector Aedes aegypti and the malaria vector Anopheles gambiae; (2) generate transgenic docking strains based on the best candidate heterospecific lox sites and determine the rate of RMCE for each using the phage P1 cre recombinase. Such an alternative system which preserves the ability to perform site-specific recombination, but avoids the integration of bacterial sequences and remains as easy to use as TE-based helper plasmids would likely be much more widely adopted, and would help to drive the field of novel genetics- based control strategies for vector-borne diseases.