Evaluation of a viral thymidine kinase gene for suicide selection in transfected mosquito cells.
An Aedes albopictus cell line previously shown to be deficient in thymidine kinase activity was transfected with a thymidine kinase gene (tk) from Herpes simplex virus. Survival of the transfected lines in a 'TK+ selective medium' indicated that the viral gene was actively expressed at a level sufficient to rescue the TK-deficient phenotype of the parent line. Unlike the parental cells, TK+ transformants (TK6:hsv cells) were sensitive to 5-bromodeoxyuridine, and contained DNA corresponding to the constructs introduced by transfection. This TK selection system will facilitate recovery of other cotransfected, non-selectable mosquito genes in cultured cells. Transformed cells were treated with several antiviral drugs to define conditions for a 'suicide selection' system, in which cells expressing the viral thymidine kinase enzyme (TK) under the control of an inducible promoter would be selectively destroyed, whereas cells expressing the endogenous mosquito enzyme would remain relatively unaffected. The anti-herpetic drug (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) showed greater cytotoxicity against transformed cells expressing the viral enzyme, and less toxicity to wild-type mosquito cells. This cell culture system provides a model for initial evaluation of suicide selection systems that may ultimately be adapted to the mosquito using sex- or tissue-specific promoters to drive expression of heterologous genes.
Mazzacano
C A
CA
Department of Entomology, University of Minnesota, St Paul 55108, USA.
Fallon
A M
AM
D000330:Aedes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D001973:Bromodeoxyuridine / Q000494:pharmacology; D002460:Cell Line; D002469:Cell Separation; D005814:Genes, Viral; D005819:Genetic Markers; D006564:Herpesviridae / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014170:Transformation, Genetic
D000998:Antiviral Agents; D005819:Genetic Markers; D011994:Recombinant Proteins; D013937:Thymidine Kinase; D001973:Bromodeoxyuridine