title: Virus-mediated expression of firefly luciferase in the parasitic protozoan Giardia lamblia.
abstract: Giardia lamblia, a prevalent human pathogen and one of the lineages that branched earliest from prokaryotes, can be infected with a double-stranded RNA virus, giardiavirus (GLV). The 6,277-bp viral genome has been previously cloned (A.L. Wang, H.-M. Yang, K.A. Shen, and C.C. Wang, Proc. Natl. Acad. Sci. USA 90:8595-8599, 1993; C.-H. Wu, C.C. Wang, H.M. Yang, and A.L. Wang, Gene, in press) and was converted to a transfection vector for G. lamblia in the present study. By flanking the firefly luciferase gene with the 5' and 3' untranslated regions (UTRs) of the GLV genome, transcript of the construct was synthesized in vitro with T7 polymerase and used to transfect G. lamblia WB trophozoites already infected with GLV (WBI). Optimal electroporation conditions used for the transfection were set at 1,000 V/cm and 500 microF, which resulted in expression of significant luciferase activity up to 120 h after electroporation. Furthermore, the mRNA and the antisense RNA of the luciferase gene were both detected by reverse transcription and PCR from 6 to 120 h postelectroporation, whereas no antisense RNA of luciferase was observed in the electroporated virus-free Giardia WB trophozoites. The mRNA of luciferase was detectable in the virus-free trophozoites by reverse transcription and PCR only up to 20 h after the electroporation, indicating that the introduced mRNA was replicated only by the viral RNA-dependent RNA polymerase inside the WBI cells. This expression of luciferase was dependent on the presence of UTRs on both ends of the viral genome transcript, including a putative packaging site that was apparently indispensable for luciferase expression. This is the first time that a viral vector in the form of mRNA URTs has been successfully used in transfecting a protozoan.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco 94143, USA.
authors.forename: D C
authors.initials: DC
authors.lastname: Yu
authors.forename: A L
authors.initials: AL
authors.lastname: Wang
authors.forename: C H
authors.initials: CH
authors.lastname: Wu
authors.forename: C C
authors.initials: CC
authors.lastname: Wang
mesh_terms: D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D001517:Coleoptera; D018274:Electroporation; D005822:Genetic Vectors; D016829:Giardia lamblia / Q000235:genetics* / Q000821:virology; D008156:Luciferases / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D012328:RNA Viruses / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D014162:Transfection
chemical_list: D012367:RNA, Viral; D008156:Luciferases