title: A PCR-based method for gene deletion and protein tagging in Trypanosoma brucei.
abstract: Sequence information on the Trypanosoma brucei genome is rapidly accumulating. As a consequence, there is a need for techniques to analyze gene function systematically. Here, we describe a polymerase chain reaction (PCR)-based method for direct gene deletion and the generation of epitope-tagged fusion proteins. The approach is based on methodologies developed for Saccharomyces cerevisiae and involves PCR amplification of a reporter cassette using primers containing flanking sequences specific to the target gene. The PCR product is then transfected directly into procyclic T. brucei cells, and homologous recombinants that carry the deleted or tagged target gene are identified.
authors.affiliation: Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA.
authors.forename: George K
authors.initials: GK
authors.lastname: Arhin
authors.forename: Shuiyuan
authors.initials: S
authors.lastname: Shen
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Ullu
authors.forename: Christian
authors.initials: C
authors.lastname: Tschudi
mesh_terms: D000818:Animals; D015153:Blotting, Western; D016133:Polymerase Chain Reaction / Q000379:methods*; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000378:metabolism