Fitness impact and stability of a transgene conferring resistance to dengue-2 virus following introgression into a genetically diverse Aedes aegypti strain.
Fitness impact and stability of a transgene conferring resistance to dengue-2 virus following introgression into a genetically diverse Aedes aegypti strain.
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DOI：
10.1371/journal.pntd.0002833
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发表时间：
2014-05
影响因子：
3.8
通讯作者：
Olson KE
中科院分区：
文献类型：
--
作者：
Franz AW;Sanchez-Vargas I;Raban RR;Black WC 4th;James AA;Olson KE
在2006年，我们报道了一个水手（Mos 1）的埃及伊蚊转化系，Carb 77，这是高度耐登革-2病毒（DENV 2）。Carb 77蚊子在摄入感染性血粉后在中肠上皮细胞中表达DENV 2特异性反向重复（IR）RNA。IR-RNA形成双链DENV 2衍生的RNA，启动细胞内抗病毒RNA干扰（RNAi）反应。然而，Carb 77蚊子在培养17代后停止表达IR-RNA，并失去了它们的DENV 2抗性表型。在当前的研究中，我们产生了具有与Carb 77相同的转基因的新的转基因品系。这些品系之一，Carb 109 M，已经遗传稳定并且对DENV 2抗性>33代。Southern杂交分析在Carb 109 M中鉴定出两个转基因整合位点。北方印迹分析检测到丰富的，瞬时表达的IR-RNA后24小时的血餐。Carb 109 M蚊子对不同的DENV 2基因型不敏感，但对其他DENV血清型不敏感。为了进一步测试适应性和稳定性，我们将Carb 109 M转基因通过回交五代并选择在每代中表达转基因的EGFP标记的个体渗入遗传多样性实验室菌株（GDLS）中。在重复回交5（BC 5）系与BC 1对照系中的转基因稳定性的比较表明，回交显著增加了转基因稳定性。我们对6个BC 5系进行了5代选择，以在最终的家族选择之前增加转基因的频率。在6个重复行中观察到的转基因频率相对于Fisher选择模型的预期的比较表明，与转基因或相关的有害基因相关的挥之不去的健身成本。尽管在最终的家系选择阶段中表现出最小的适合度损失（相对于GDLS），但我们能够在一个家系Carb 109 M/GDLS.BC5.HZ中选择转基因的纯合子。这个家族已经遗传稳定并且DENV 2难治了多代。Carb 109 M/GDLS.BC5.HZ代表了用于测试原理验证载体群体替换的重要品系。DENV 2序列衍生的IR RNA在蚊子中肠中的表达启动了抗病毒细胞内RNAi反应，其有效地阻断DENV 2感染并深刻地削弱了埃及伊蚊中该病毒的载体能力。Carb 109 M菌株中DENV 2特异性IR RNA表达在实验室培养中维持了33代基于RNAi的难治表型。这两个转基因整合位点在多代后和渗入到遗传多样性（GDLS）Ae中后是稳定的。埃及人口。转基因渗入GDLS遗传背景将GDLS从DENV 2易感表型改变为DENV 2难治表型。DENV 2难治性纯合系Carb 109 M/GDLS.BC5.HZ表现出（相对于GDLS）与转基因相关的最小适合度损失。这条线可能是一个潜在的候选人证明的原则领域的研究。
In 2006, we reported a mariner (Mos1)-transformed Aedes aegypti line, Carb77, which was highly resistant to dengue-2 virus (DENV2). Carb77 mosquitoes expressed a DENV2-specific inverted-repeat (IR) RNA in midgut epithelial cells after ingesting an infectious bloodmeal. The IR-RNA formed double-stranded DENV2-derived RNA, initiating an intracellular antiviral RNA interference (RNAi) response. However, Carb77 mosquitoes stopped expressing the IR-RNA after 17 generations in culture and lost their DENV2-refractory phenotype. In the current study, we generated new transgenic lines having the identical transgene as Carb77. One of these lines, Carb109M, has been genetically stable and refractory to DENV2 for >33 generations. Southern blot analysis identified two transgene integration sites in Carb109M. Northern blot analysis detected abundant, transient expression of the IR-RNA 24 h after a bloodmeal. Carb109M mosquitoes were refractory to different DENV2 genotypes but not to other DENV serotypes. To further test fitness and stability, we introgressed the Carb109M transgene into a genetically diverse laboratory strain (GDLS) by backcrossing for five generations and selecting individuals expressing the transgene's EGFP marker in each generation. Comparison of transgene stability in replicate backcross 5 (BC5) lines versus BC1 control lines demonstrated that backcrossing dramatically increased transgene stability. We subjected six BC5 lines to five generations of selection based on EGFP marker expression to increase the frequency of the transgene prior to final family selection. Comparison of the observed transgene frequencies in the six replicate lines relative to expectations from Fisher's selection model demonstrated lingering fitness costs associated with either the transgene or linked deleterious genes. Although minimal fitness loss (relative to GDLS) was manifest in the final family selection stage, we were able to select homozygotes for the transgene in one family, Carb109M/GDLS.BC5.HZ. This family has been genetically stable and DENV2 refractory for multiple generations. Carb109M/GDLS.BC5.HZ represents an important line for testing proof-of-principle vector population replacement. Expression of a DENV2 sequence-derived IR RNA in the mosquito midgut initiates an antiviral intracellular RNAi response that efficiently blocks DENV2 infection and profoundly impairs vector competence for that virus in Aedes aegypti. DENV2-specific IR RNA expression in the Carb109M strain has maintained the RNAi-based, refractory phenotype for 33 generations in laboratory culture. The two transgene integration sites were stable after multiple generations and following introgression into a genetically-diverse (GDLS) Ae. aegypti population. Introgression of the transgene into the GDLS genetic background changed GDLS from a DENV2 susceptible phenotype to a DENV2 refractory phenotype. The DENV2 refractory homozygous line, Carb109M/GDLS.BC5.HZ, exhibits (relative to GDLS) minimal fitness loss associated with the transgene. This line could be a potential candidate for proof-of-principle field studies.
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影响因子：
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