Efficient transformation of the yellow fever mosquito Aedes aegypti using the piggyBac transposable element vector pBac[3xP3-EGFP afm]
Efficient transformation of the yellow fever mosquito Aedes aegypti using the piggyBac transposable element vector pBac[3xP3-EGFP afm]
复制标题
DOI：
10.1016/s0965-1748(01)00120-5
复制
发表时间：
2001-11-01
影响因子：
3.8
通讯作者：
Raikhel, AS
中科院分区：
文献类型：
--
作者：
Kokoza, V;Ahmed, A;Raikhel, AS
我们报道了利用iggyBac转座元件载体pBAC[3xP3-EGFP AFM]在黄热病蚊子埃及伊蚊中实现了有效的种系转化。建立并鉴定了两个转基因株系，每个转基因株系均含有由人工3xP3启动子调控的眼睛特异表达增强型绿色荧光蛋白(EGFP)标记基因的VG-防御素A转基因植株。基因组DNA的Southern杂交和反向PCR分析表明，pBAC[3xP3-EGFP AFM，VG-DefA]转座子在每个转基因株系中都有精确的单拷贝插入。对于每个品系，遗传分析证实了在G(2)-G(6)世代中蚊子基因组中整个转座子结构的稳定性和完整性。纯合子转基因品系的成功建立表明，在这两种情况下，转座子都发生了与蚊子基因组的非致命性整合。3xP3-EGFP标记在不同遗传背景的蚊子中进行了测试。在白眼转基因蚊子中，从新孵化的一龄幼虫到成虫，在整个发育阶段都观察到了强烈的眼睛特异性GFP表达。将3xP3-EGFP转化子与转朱砂基因的kh(W)白眼蚊杂交，在红眼蚊子中观察到类似的荧光水平和模式。重要的是，3xP3-EGFP白眼转化子和野生型Rockefeller/Uga品系的黑眼杂交种幼虫和蛹阶段都有强烈的眼睛特异性GFP表达，证明了3xP3-EGFP作为野生型蚊子转化标记基因的实用性。最后，对VG-DefA转基因在两个已建立的品系中的表达进行了分析，结果表明VG-DefA基因具有很强的血粉活性和受VG 1.8-kb 5‘上游区域调控的脂肪体特异性表达。(C)2001爱思唯尔科学有限公司。保留所有权利。
We report efficient germ-line transformation in the yellow fever mosquito Aedes aegypti accomplished using the piggyBac transposable element vector pBac[3xP3-EGFP afm]. Two,transgenic lines were established and characterized; each contained the Vg-Defensin A transgene with strong eye-specific expression of the enhanced green fluorescent protein (EGFP) marker gene regulated by the artificial 3xP3 promoter. Southern blot hybridization and inverse PCR analyses of genomic DNA demonstrated a precise piggyBac-mediated, single copy insertion of the pBac[3xP3-EGFP afm,Vg-DefA] transposon in each transgenic line. For each line, genetic analysis confirmed stability and integrity of the entire transposon construct in the mosquito genome through the G(2)-G(6) generations. Successful establishment of homozygous transgenic lines indicated that in both cases a non-lethal integration of the transposon into the mosquito genome had occurred. The 3xP3-EGFP marker was tested in mosquitoes with different genetic backgrounds. In white-eyed transgenic mosquitoes, the strong eye-specific expression of GFP was observed throughout all stages of development, starting from newly hatched first instar larvae to adults. A similar level and pattern of fluorescence was observed in red-eyed mosquitoes that were generated by crossing the 3xP3-EGFP transformants with the kh(w) white-eye mosquitoes transformed with the Drosophila cinnabar gene. Importantly, the utility of the 3xP3-EGFP, as marker gene for transformation of wild type mosquitoes, was demonstrated by strong eye-specific GFP expression in larval and pupal stages of black-eyed hybrids of the 3xP3-EGFP white-eye transformants and the wild type Rockefeller/UGAL strain. Finally, analysis of the Vg-DefA transgene expression in transformants from two established lines demonstrated strong blood-meal activation and fat-body-specific expression regulated by the Vg 1.8-kb 5' upstream region. (C) 2001 Elsevier Science Ltd. All rights reserved.