Naegleria fowleri: Functional expression of the Nfa1 protein in transfected Naegleria gruberi by promoter modification

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 vec...

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Bibliographic Details
Published in:Experimental parasitology 2006-02, Vol.112 (2), p.115-120
Main Authors: Song, Kyoung-Ju, Jeong, Seok-Ryoul, Park, Sun, Kim, Kyongmin, Kwon, Myung-Hee, Im, Kyung-Il, Pak, Jhang Ho, Shin, Ho-Joon
Format: Article
Language:English
Subjects:
Actins - genetics
Animals
Antibodies, Protozoan - immunology
Antigens, Protozoan - biosynthesis
Antigens, Protozoan - genetics
Antigens, Protozoan - immunology
Base Sequence
Biological and medical sciences
Blotting, Western
CHO Cells
Cricetinae
Cricetulus
Cytomegalovirus
Fundamental and applied biological sciences. Psychology
Gene Expression Regulation - physiology
Genetic Vectors
Green Fluorescent Proteins - genetics
Immunofluorescent assay
Life cycle. Host-agent relationship. Pathogenesis
Mice
Mice, Inbred BALB C
Microscopy, Fluorescence
Molecular Sequence Data
Naegleria - genetics
Naegleria - metabolism
Naegleria fowleri
Naegleria gruberi
Plasmids
Promoter
Promoter Regions, Genetic - genetics
Protozoa
Protozoan Proteins - biosynthesis
Protozoan Proteins - genetics
Protozoan Proteins - immunology
Reverse Transcriptase Polymerase Chain Reaction
Transfection
Transient transfection
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Summary: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.
ISSN:0014-4894
1090-2449
DOI:10.1016/j.exppara.2005.10.004