title: Stable transfection of Acanthamoeba.
abstract: The promoter activity of an Acanthamoeba polyubiquitin gene was analyzed in its homologous system. A modified calcium phosphate transfection method using a neomycin marker vector was developed to achieve highly efficient transfection of the Acanthamoeba polyubiquitin gene into Acanthamoeba cells. In this transfection procedure, the calcium phosphate-DNA complex was formed gradually in the medium during incubation with cells and precipitated on the cells. The crucial factors for obtaining efficient transfection were the pH (6.95) of the transfection buffer used for the calcium phosphate precipitation and the amount (25 micrograms/96-well tissue culture plate) and form (circular) of transfecting DNA. Under these conditions, Acanthamoeba isolate 1B6 was transfected at an efficiency of about 40% with the constructed vector pOPSBU, a pOP13CAT-based polyubiquitin gene incorporated neomycin resistance vector. Acanthamoeba polyphaga was transfected at an efficiency of about 10% with this vector. Transfection of both Acanthamoeba strains appeared to result in low copy plasmid integration (about two copies per cell are suggested). The chloramphenicol acetyltransferase (CAT) assays showed that the promoter of the Acanthamoeba polyubiquitin gene in the constructed vector was especially strong in A. polyphaga, thus the pOPSBU-Acanthamoeba system may be useful for the construction of cDNA expression libraries, as well as for the expression of cloned genes.
authors.affiliation: Department of Biology, University of Houston, TX 77204-5513, USA.
authors.forename: J
authors.initials: J
authors.lastname: Yin
authors.forename: H R
authors.initials: HR
authors.lastname: Henney
mesh_terms: D000048:Acanthamoeba / Q000235:genetics*; D000818:Animals; D001704:Biopolymers / Q000235:genetics*; D015139:Blotting, Southern; D002130:Calcium Phosphates / Q000494:pharmacology; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D003001:Cloning, Molecular; D004270:DNA, Circular / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009355:Neomycin / Q000494:pharmacology; D025821:Polyubiquitin; D011401:Promoter Regions, Genetic; D014162:Transfection; D014452:Ubiquitins / Q000235:genetics*
chemical_list: D001704:Biopolymers; D002130:Calcium Phosphates; D004270:DNA, Circular; D014452:Ubiquitins; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; D025821:Polyubiquitin; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; D015500:Chloramphenicol O-Acetyltransferase; D009355:Neomycin; C485829:calcium phosphate, dibasic, anhydrous