A PCR-based method for gene deletion and protein tagging in Trypanosoma brucei.
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.
Arhin
George K
GK
Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA.
Shen
Shuiyuan
S
Ullu
Elisabetta
E
Tschudi
Christian
C
D000818:Animals; D015153:Blotting, Western; D016133:Polymerase Chain Reaction / Q000379:methods*; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000378:metabolism