Abstract
IntroductionGenetic 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.Results 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.ConclusionsThese new promoters will be useful for driving expression of genes of interest in transgenic Cx. tarsalis and perhaps other insects.
Highlights
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
An essential step in this process is to identify suitable stage- and tissue-specific promoters to drive expression of antipathogen genes of interest, work on which has been primarily focused on midgut, salivary gland and fatbody promoters of Anopheles and Aedes mosquitoes
The transcription factor binding sites (GATA, C/EBP, EcRE) which are required for tissue- and stage-specific vitellogenin expression in mosquito vitellogenin genes are usually located approximately 2 kb 59 upstream of the initial vitellogenin transcriptional start site [1]
Summary
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. The promoter regions of the Anopheles gambiae carboxypeptidase (AgCP) and Aedes aegypti carboxypeptidase (AeCP) genes have been isolated and shown to drive midgut-specific bloodmeal-inducible gene expression [2]. The promoter fragments of An. gambiae female salivary gland-specific genes AgApy and D7r4 directed expression of a LacZ reporter gene in the An. stephensi salivary gland [4]. A female salivary gland-specific promoter for a gene encoding anopheline antiplatelet protein (AAPP) isolated from An. stephensi was able to drive expression of DsRed in the salivary glands [5]. A 1.7 kb promoter fragment of An. gambiae vitellogenin gene (VgT2) was able to direct expression of green fluorescent protein (GFP) in a tissue-, stage-, and sex-specific manner in transgenic An. stephensi [7]
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