title: Restriction enzyme-mediated integration elevates transformation frequency and enables co-transfection of Toxoplasma gondii.
abstract: This report describes the use of restriction enzyme-mediated integration (REMI) to increase the transformation frequency and allow co-transfection of several unselected constructs under the selection of a single selectable marker. We found that while BamHI (the enzyme used to originally demonstrate REMI (Schiestl, R.H. and Petes, T.D. (1991) Integration of DNA fragments by illegitimate recombination in Saccharomyces cerevisiae. Proc. Nati. Acad. Sci. USA 88, 7585-7589) increased the number of transformants by 2-5-fold over the control without added enzyme, NotI proved to be a further 29-46-times more effective in enhancing stable transformation. This simple technique was used in the transformation of three non-selective markers (two modified membrane proteins and beta-galactosidase) with a selectable construct expressing chloramphenicol acetyltransferase. Following chloramphenicol selection, four out of ten independent transformants stably acquired all four constructs with at least two expressing all four genes at the protein level. These results demonstrate that REMI may be used in the efficient stable transformation and co-transfection of this and perhaps other protozoan parasites.
authors.affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, CA, 94305-5402, USA.
authors.forename: M
authors.initials: M
authors.lastname: Black
authors.forename: F
authors.initials: F
authors.lastname: Seeber
authors.forename: D
authors.initials: D
authors.lastname: Soldati
authors.forename: K
authors.initials: K
authors.lastname: Kim
authors.forename: J C
authors.initials: JC
authors.lastname: Boothroyd
mesh_terms: D000818:Animals; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D015245:Deoxyribonuclease BamHI; D015252:Deoxyribonucleases, Type II Site-Specific; D005819:Genetic Markers; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014122:Toxoplasma / Q000201:enzymology / Q000235:genetics*; D014162:Transfection; D014170:Transformation, Genetic; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan; D005819:Genetic Markers; D015500:Chloramphenicol O-Acetyltransferase; D015245:Deoxyribonuclease BamHI; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases; D001616:beta-Galactosidase