title: Stable transfection of Acanthamoeba castellanii.
abstract: A simple method for stable transfection of Acanthamoeba castellanii using plasmids which confer resistance to neomycin G418 is described. Expression of neomycin phosphotransferase is driven by the Acanthamoeba TBP gene promoter, and can be monitored by cell growth in the presence of neomycin G418 or by Western blot analysis. Transfected cells can be passaged in the same manner as control cells and can be induced to differentiate into cysts, in which form they maintain resistance to neomycin G418 for at least several weeks, although expression of neomycin phosphotransferase is repressed during encystment. Expression of EGFP or an HA-tagged EGFP-TBP fusion can be driven from the same plasmid, using an additional copy of the Acanthamoeba TBP gene promoter or a deletion mutant. The TBP-EGFP fusion is localized to the nucleus, except in a small proportion of presumptive pre-mitotic cells. EGFP expression can also be driven by the cyst-specific CSP21 gene promoter, which is completely repressed in growing cells but strongly induced in differentiating cells. Transfected cells maintain their phenotype for several weeks, even in the absence of neomycin G418, suggesting that transfected genes are stably integrated within the genome. These results demonstrate the utility of the neomycin resistance based plasmids for stable transfection of Acanthamoeba, and may assist a number of investigations.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Vermont, Burlington VT 05405, USA.
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Peng
authors.forename: Romaica
authors.initials: R
authors.lastname: Omaruddin
authors.forename: Erik
authors.initials: E
authors.lastname: Bateman
mesh_terms: D048248:Acanthamoeba castellanii / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D002454:Cell Differentiation; D017353:Gene Deletion; D018507:Gene Expression Regulation, Developmental; D005821:Genetic Techniques; D016678:Genome; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008938:Mitosis; D009355:Neomycin / Q000494:pharmacology; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D012319:RNA Polymerase II; D009355:Neomycin