title: Naegleria fowleri: functional expression of the Nfa1 protein in transfected Naegleria gruberi by promoter modification.
abstract: To establish a transient transfection system in a Naegleria, we constructed three nfa1-pEGFP-N1 vectors by the promoter replacement and insertion of a nfa1 gene and transfected the DNAs into Naegleria gruberi using a lipid reagent. The transfection efficiency and usefulness of the three modified vectors were estimated by identifying the expressions of the EGFP and Nfa1 protein from N. gruberi. After transfection, the Nfa1 protein was functionally expressed on pseudopodia of N. gruberi. The strong GFP fluorescence was observed in N. gruberi transfected with the actin-nfa1-pEGFP-N1 vector, of which the CMV promoter region in the expression vector was replaced with the actin 5' UTR region. Additionally, when transgenic N. gruberi trophozoites were co-cultured with CHO target cells, the Nfa1 protein was also located on cytoplasm and pseudopodia, especially on a food cup that was formed in contact with target cells as it shown in pathogenic N. fowleri.
authors.affiliation: Department of Microbiology, Ajou University School of Medicine, Suwon 443-721, Republic of Korea.
authors.forename: Kyoung-Ju
authors.initials: KJ
authors.lastname: Song
authors.forename: Seok-Ryoul
authors.initials: SR
authors.lastname: Jeong
authors.forename: Sun
authors.initials: S
authors.lastname: Park
authors.forename: Kyongmin
authors.initials: K
authors.lastname: Kim
authors.forename: Myung-Hee
authors.initials: MH
authors.lastname: Kwon
authors.forename: Kyung-Il
authors.initials: KI
authors.lastname: Im
authors.forename: Jhang Ho
authors.initials: JH
authors.lastname: Pak
authors.forename: Ho-Joon
authors.initials: HJ
authors.lastname: Shin
mesh_terms: D000199:Actins / Q000235:genetics; D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology; D000953:Antigens, Protozoan / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005786:Gene Expression Regulation / Q000502:physiology*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009253:Naegleria / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics*; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: D000199:Actins; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; C460560:Nfa1 protein, Naegleria fowleri; D015800:Protozoan Proteins; D049452:Green Fluorescent Proteins