title: Detection of expressed chloramphenicol acetyltransferase in the saliva of Culex pipiens mosquitoes.
abstract: Mosquito salivary glands play an important role in the transmission of arthropod-borne pathogens. The ability to express genes in mosquitoes would be a powerful approach to characterize salivary gland genes, and to reveal important vector determinants of pathogen transmission. Here we report the use of a double subgenomic Sindbis (dsSIN) virus, designated TE/3'2J/CAT, and a packaged Sindbis replicon virus, designated rep5/CAT/26S, to express chloramphenicol acetyltransferase (CAT) protein in the salivary glands and saliva of transduced female Culex pipiens pipiens. Indirect immunofluorescence analysis revealed that salivary glands of these mosquitoes infected with either TE/3'2J/CAT or rep5/CAT/26S virus (4 or 6 days post-infection (p.i.)) were positive for both SIN E1 antigen and CAT protein. Saliva collected from mosquitoes transduced with TE/3'2J/CAT virus contained a unique 25 kDa protein that corresponded to the size of CAT protein. Additionally, CAT activity assays revealed that saliva collected from mosquitoes transduced with either TE/3'2J/CAT or rep5/CAT/26S virus could contain greater than 5.0 x 10(-5) units of CAT enzyme (3.0 x 10(6) CAT trimers).
authors.affiliation: Department of Microbiology, Colorado State University, Fort Collins 80523, USA.
authors.forename: K I
authors.initials: KI
authors.lastname: Kamrud
authors.forename: K E
authors.initials: KE
authors.lastname: Olson
authors.forename: S
authors.initials: S
authors.lastname: Higgs
authors.forename: A M
authors.initials: AM
authors.lastname: Powers
authors.forename: J O
authors.initials: JO
authors.lastname: Carlson
authors.forename: B J
authors.initials: BJ
authors.lastname: Beaty
mesh_terms: D000818:Animals; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D006224:Cricetinae; D003465:Culex / Q000378:metabolism*; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D005822:Genetic Vectors; D008562:Membrane Glycoproteins / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012463:Saliva / Q000378:metabolism; D012469:Salivary Glands / Q000378:metabolism; D012845:Sindbis Virus / Q000235:genetics*; D014759:Viral Envelope Proteins / Q000235:genetics*
chemical_list: D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C022012:glycoprotein E1, Sindbis virus; D015500:Chloramphenicol O-Acetyltransferase