Regulated expression of microinjected DNA in adult Aedes aegypti mosquitoes.
We have developed a novel molecular genetic approach to investigating gene regulation in adult mosquitoes called whole body transfection (WBT). This DNA microinjection method allows for both constitutive and regulated expression of plasmid vectors in the fat body and midgut of adult mosquitoes within 24 h of injection. Using a luciferase reporter gene containing the Aedes aegypti heat shock protein 70 (Hsp70) promoter, we optimized the WBT protocol at various times post-injection and used these parameters to measure the expression of a vitellogenin-luciferase reporter gene in response to blood meal feeding. These studies showed that a 843 bp fragment of the Ae. aegypti vitellogenin-C (VgC) promoter caused a greater than 200-fold induction of luciferase activity in a strict tissue-specific manner, and only in response to feeding. Functional mapping of the VgC promoter by WBT identified essential upstream regulatory elements in the region spanning -780 to -182 bp from the transcriptional start site. We also constructed a lipopolysaccharide-regulated expression vector using a 1096 bp genomic fragment of the Ae. aegypti cecropin B (CecB) promoter. Our data show that four days after WBT injection, the CecB-luciferase reporter gene could be induced more than 100-fold in the fat body following lipopolysaccharide injection. Moreover, we found that lipopolysaccharide-induction of the CecB reporter gene occurred up to 28 days post-WBT injection. These data suggest that WBT could provide a novel strategy to express recombinant proteins and RNAi constructs in adult mosquitoes using conventional microinjection methods.
Isoe
J
J
Department of Biochemistry and Molecular Biophysics, The University of Arizona, Tucson, AZ 85721, USA.
Kunz
S
S
Manhart
C
C
Wells
M A
MA
Miesfeld
R L
RL
D000330:Aedes / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000008:administration & dosage* / Q000235:genetics*; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism; D011401:Promoter Regions, Genetic
D018840:HSP70 Heat-Shock Proteins; D019476:Insect Proteins; D008070:Lipopolysaccharides; C031162:cecropin B protein, Insecta; D004247:DNA