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 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.
Song
Kyoung-Ju
KJ
Department of Microbiology, Ajou University School of Medicine, Suwon 443-721, Republic of Korea.
Jeong
Seok-Ryoul
SR
Park
Sun
S
Kim
Kyongmin
K
Kwon
Myung-Hee
MH
Im
Kyung-Il
KI
Pak
Jhang Ho
JH
Shin
Ho-Joon
HJ
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
D000199:Actins; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; C460560:Nfa1 protein, Naegleria fowleri; D015800:Protozoan Proteins; D049452:Green Fluorescent Proteins