Inhibition of luciferase expression in transgenic Aedes aegypti mosquitoes by Sindbis virus expression of antisense luciferase RNA.
A rapid and reproducible method of inhibiting the expression of specific genes in mosquitoes should further our understanding of gene function and may lead to the identification of mosquito genes that determine vector competence or are involved in pathogen transmission. We hypothesized that the virus expression system based on the mosquito-borne Alphavirus, Sindbis (Togaviridae), may efficiently transcribe effector RNAs that inhibit expression of a targeted mosquito gene. To test this hypothesis, germ-line-transformed Aedes aegypti that express luciferase (LUC) from the mosquito Apyrase promoter were intrathoracically inoculated with a double subgenomic Sindbis (dsSIN) virus TE/3'2J/anti-luc (Anti-luc) that transcribes RNA complementary to the 5' end of the LUC mRNA. LUC activity was monitored in mosquitoes infected with either Anti-luc or control dsSIN viruses expressing unrelated antisense RNAs. Mosquitoes infected with Anti-luc virus exhibited 90% reduction in LUC compared with uninfected and control dsSIN-infected mosquitoes at 5 and 9 days postinoculation. We demonstrate that a gene expressed from the mosquito genome can be inhibited by using an antisense strategy. The dsSIN antisense RNA expression system is an important tool for studying gene function in vivo.
Johnson
B W
BW
Arthropod-Borne and Infectious Diseases Laboratory (AIDL), Department of Microbiology, Colorado State University, Fort Collins, CO 80523, USA.
Olson
K E
KE
Allen-Miura
T
T
Rayms-Keller
A
A
Carlson
J O
JO
Coates
C J
CJ
Jasinskiene
N
N
James
A A
AA
Beaty
B J
BJ
Higgs
S
S
D000330:Aedes / Q000201:enzymology*; D000818:Animals; D030801:Animals, Genetically Modified; D001081:Apyrase / Q000378:metabolism; D015153:Blotting, Western; D008156:Luciferases / Q000235:genetics*; D016372:RNA, Antisense / Q000235:genetics*; D015203:Reproducibility of Results; D012469:Salivary Glands / Q000201:enzymology / Q000821:virology; D012845:Sindbis Virus / Q000235:genetics*
D016372:RNA, Antisense; D008156:Luciferases; D001081:Apyrase