Stable transformation of trypanosomatids through targeted chromosomal integration of the selectable marker gene encoding blasticidin S deaminase

The susceptibilities of the protozoan parasites Leishmania mexicana and Try panosoma brucei to the nucleoside antibiotic blasticidin S were assessed. A concentration of 10 mu g ml(-1) was sufficient to cause cell death within 72 h of L. mexicana promastigotes and bloodstream forms of T. brucei in vit ro. The gene encoding blasticidin S deaminase (BSD) was therefore incorpora ted into cassettes for targeting to the cysteine proteinase C locus of L. m exicana (CPC::BSD) and the tubulin locus of T, brucei (tub.. RAD51-BSR). Fo llowing transfection of mutant parasites that contained other well-establis hed selectable marker genes (HYG, NEO, BLE, PAC and SAT), clones resistant to 10 mu g ml(-1) blasticidin S were shown by PCR and Southern blotting to have integrated the cassettes by homologous recombination. The results conf irm that BSD can be used as a selectable marker gene for targeted chromosom al integration during genetic manipulations of trypanosomatids, (C) 2000 Fe deration of European Microbiological Societies. Published by Elsevier Scien ce B.V. All rights reserved.