A transgenic sensor strain for monitoring the RNAi pathway in the yellow fever mosquito, Aedes aegypti
A transgenic sensor strain for monitoring the RNAi pathway in the yellow fever mosquito, Aedes aegypti
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DOI：
10.1016/j.ibmb.2008.04.002
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发表时间：
2008-07-01
影响因子：
3.8
通讯作者：
Myles, Kevin M.
中科院分区：
文献类型：
--
作者：
Adelman, Zach N.;Anderson, Michelle A. E.;Myles, Kevin M.
RNA干扰途径在无脊椎动物中起抗病毒防御的作用。为了产生“感测”埃及伊蚊中RNAi途径的状态的表型标记，开发转基因菌株以在眼中表达EGFP和DsRED标记基因，以及与EGFP基因的一部分同源的双链RNA。转基因“传感器”蚊子表现出强烈的眼睛特异性DsRED表达，几乎没有EGFP，表明基于RNAi的沉默。小RNA的克隆和高通量测序证实，转基因的反向重复序列被成功地加工成短干扰RNA的蚊子RNAi途径。当A.基因DCR-2或AGO-2的埃及伊蚊同源物被敲低，在蚊子眼睛中观察到EGFP荧光的明显增加。通过北方印迹和实时PCR，DCR-2的敲低也与EGFP mRNA水平的增加相关。AGO-3是一种参与生殖细胞特异性皮尔纳途径的基因，敲除AGO-3后，EGFP的表达在mRNA或蛋白质水平均未恢复。这种转基因传感器菌株现在可以用于鉴定蚊子RNAi途径的其他组分，并有可能用于鉴定RNAi的虫媒病毒抑制子。(C)2008爱思唯尔有限公司版权所有。
The RNA interference pathway functions as an antiviral defense in invertebrates. In order to generate a phenotypic marker which "senses" the status of the RNAi pathway in Aedes aegypti, transgenic strains were developed to express EGFP and DsRED marker genes in the eye, as well as double-stranded RNA homologous to a portion of the EGFP gene. Transgenic "sensor" mosquitoes exhibited robust eye-specific DsRED expression with little EGFP, indicating RNAi-based silencing. Cloning and high-throughput sequencing of small RNAs confirmed that the inverted-repeat transgene was successfully Processed into short-interfering RNAs by the mosquito RNAi pathway. When the A. aegypti homologues of the genes DCR-2 or AGO-2 were knocked down, a clear increase in EGFP fluorescence was observed in the mosquito eyes. Knockdown of DCR-2 was also associated with an increase in EGFP mRNA levels, as by Northern blot and real-time PCR. Knockdown of AGO-3, a gene involved in the germline-specific piRNA pathway, did not restore EGFP expression at either the mRNA or protein level. This transgenic sensor strain can now be used to identify other components of the mosquito RNAi pathway and has the potential to be used in the identification of arboviral suppressors of RNAi. (C) 2008 Elsevier Ltd. All rights reserved.