Chen Song, Rasgon Jason L
The W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, United States of America.
Department of Entomology, The Center for Infectious Disease Dynamics and the Huck Institutes of The Life Sciences, Millennium Science Complex, The Pennsylvania State University, University Park, Pennsylvania, United States of America.
PLoS One. 2014 Feb 25;9(2):e88994. doi: 10.1371/journal.pone.0088994. eCollection 2014.
Genetic modification, or transgenesis, is a powerful technique to investigate the molecular interactions between vector-borne pathogens and their arthropod hosts, as well as a potential novel approach for vector-borne disease control. Transgenesis requires the use of specific regulatory regions, or promoters, to drive expression of genes of interest in desired target tissues. In mosquitoes, the vast majority of described promoters are from Anopheles and Aedes mosquitoes.
Culex tarsalis is one of the most important vectors of arboviruses (including West Nile virus) in North America, yet it has not been the subject of molecular genetic study. In order to facilitate molecular genetic work in this important vector species, we isolated four fat body-specific promoter sequences located upstream of the Cx. tarsalis vitellogenin genes (Vg1a, Vg1b, Vg2a and Vg2b). Sequences were analyzed in silico to identify requisite cis-acting elements. The ability for promoter sequences to drive expression of green fluorescent protein (GFP) in vivo was investigated using transgenic Drosophila melanogaster. All four promoters were able to drive GFP expression but there was dramatic variation between promoters and between individual Drosophila lines, indicating significant position effects. The highest expression was observed in line Vg2bL3, which was >300-fold higher than the lowest line Vg1aL2.
These new promoters will be useful for driving expression of genes of interest in transgenic Cx. tarsalis and perhaps other insects.
基因改造，即转基因技术，是一种用于研究媒介传播病原体与其节肢动物宿主之间分子相互作用的强大技术，也是控制媒介传播疾病的一种潜在新方法。转基因需要使用特定的调控区域或启动子，以驱动目标基因在所需靶组织中的表达。在蚊子中，绝大多数已描述的启动子来自按蚊和伊蚊。
致倦库蚊是北美最重要的虫媒病毒（包括西尼罗河病毒）传播媒介之一，但尚未成为分子遗传学研究的对象。为了促进对这一重要媒介物种的分子遗传学研究，我们分离了位于致倦库蚊卵黄原蛋白基因（Vg1a、Vg1b、Vg2a和Vg2b）上游的四个脂肪体特异性启动子序列。对序列进行了电子分析，以确定必需的顺式作用元件。使用转基因黑腹果蝇研究了启动子序列在体内驱动绿色荧光蛋白（GFP）表达的能力。所有四个启动子都能够驱动GFP表达，但启动子之间以及果蝇个体品系之间存在显著差异，表明存在明显的位置效应。在品系Vg2bL3中观察到最高表达，比最低品系Vg1aL2高300倍以上。
这些新的启动子将有助于在转基因致倦库蚊以及可能的其他昆虫中驱动目标基因的表达。
