title: Amplification, expression, and packaging of a foreign gene by giardiavirus in Giardia lamblia.
abstract: Giardia lamblia is an intestinal protozoan parasite and one of the earliest eukaryotic divergents. The trophozoite multiplies via asexual binary fission and lacks all natural means of lateral gene transfer. A system is developed here for long-term expression of a foreign gene in this organism by exploiting recombinant virions derived from the giardiavirus (GLV), a double-stranded RNA virus that infects many Giardia isolates. An in vitro transcript of the cloned GLV cDNA, comprising the firefly luciferase-encoding region flanked by 5' and 3' fragments of GLV positive-strand RNA, was electroporated into GLV-infected trophozoites. Luciferase activity in electroporated cells peaked on day 2 at levels 6 orders of magnitude above background. Expression of this foreign gene remained at 80% of its peak level after 30 days in the absence of selective pressure. The chimeric RNA was replicated as double-stranded RNA and packaged into virus-like particles. The recombinant virions were partially purified from the wild-type helper virus by CsCl equilibrium density-gradient centrifugation and used to superinfect Giardia trophozoites. At multiplicities of infection of 100 or higher, these chimeric virions were able to initiate new rounds of expression of luciferase activity in the superinfected cells. Thus, the engineered virion can be successfully used to introduce and efficiently express a heterologous gene in this eukaryotic microorganism.
authors.affiliation: Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446, USA.
authors.forename: D C
authors.initials: DC
authors.lastname: Yu
authors.forename: A L
authors.initials: AL
authors.lastname: Wang
authors.forename: C C
authors.initials: CC
authors.lastname: Wang
mesh_terms: D000818:Animals; D001483:Base Sequence; D004279:DNA, Viral; D005784:Gene Amplification; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D016829:Giardia lamblia / Q000378:metabolism* / Q000821:virology; D019198:Giardiavirus / Q000235:genetics* / Q000502:physiology; D008156:Luciferases / Q000235:genetics*; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D019065:Virus Assembly
chemical_list: D004279:DNA, Viral; D008156:Luciferases