title: Expression of an antisense dihydrofolate reductase transcript in transfected mosquito cells: effects on growth and plating efficiency.
abstract: Novel approaches to control of vector-borne disease include potential use of transgenic insects, in which molecular mechanisms will be induced to prevent transmission of pathogenic organisms. The infrastructure essential to this technology includes the cloning of essential genes from vector insects, and the development of efficient transformation strategies. In this study, we use a continuous mosquito (Aedes albopictus) cell line and a cloned mosquito dihydrofolate reductase gene to demonstrate a transgenic approach that may be used to select for the presence or absence of particular gene functions in transfected cells. Plasmids containing the dihydrofolate reductase gene in sense and antisense orientation, under the regulation of a temperature-inducible promoter, were expressed in stably transfected mosquito cells. At the normal growth temperature of 28 degrees C, or after mild heat induction at 34 degrees C, expression of the dihydrofolate reductase construct in sense orientation had little effect on cell growth. In contrast, recovery of clones transfected with the antisense construct was reduced, and induction of antisense transcripts at 34 degrees C further compromised cell growth and viability. Clones transfected with the sense construct retained significantly higher copy numbers of foreign DNA than did cells transfected with the antisense construct. These studies provide a basis for use of sense and antisense dihydrofolate reductase constructs to recover transfected mosquito cells with specific desired phenotypes, based on the relative expression of cloned genes of interest.
authors.affiliation: Department of Entomology, University of Minnesota, St. Paul.
authors.forename: F A
authors.initials: FA
authors.lastname: Shotkoski
authors.forename: A M
authors.initials: AM
authors.lastname: Fallon
mesh_terms: D000330:Aedes / Q000166:cytology / Q000201:enzymology / Q000235:genetics*; D000818:Animals; D030801:Animals, Genetically Modified; D002455:Cell Division; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis; D015971:Gene Expression Regulation, Enzymologic; D006360:Heat-Shock Proteins / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D016372:RNA, Antisense / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D015183:Restriction Mapping; D013696:Temperature; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D006360:Heat-Shock Proteins; D016372:RNA, Antisense; D012333:RNA, Messenger; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase