Open AccessStable transformation of trypanosomatids through targeted chromosomal integration of the selectable marker gene encoding blasticidin S deaminase
Author(s) -
Brooks Darren R,
McCulloch Richard,
Coombs Graham H,
Mottram Jeremy C
Publication year - 2000
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2000.tb09119.x
Subject(s) - selectable marker , biology , trypanosoma brucei , gene , cytidine deaminase , locus (genetics) , mutant , transformation (genetics) , southern blot , microbiology and biotechnology , genetics
Abstract The susceptibilities of the protozoan parasites Leishmania mexicana and Trypanosoma brucei to the nucleoside antibiotic blasticidin S were assessed. A concentration of 10 μg ml −1 was sufficient to cause cell death within 72 h of L. mexicana promastigotes and bloodstream forms of T. brucei in vitro. The gene encoding blasticidin S deaminase ( BSD ) was therefore incorporated into cassettes for targeting to the cysteine proteinase C locus of L. mexicana ( CPC::BSD ) and the tubulin locus of T. brucei ( tub::RAD51‐BSR ). Following transfection of mutant parasites that contained other well‐established selectable marker genes ( HYG, NEO, BLE, PAC and SAT ), clones resistant to 10 μg ml −1 blasticidin S were shown by PCR and Southern blotting to have integrated the cassettes by homologous recombination. The results confirm that BSD can be used as a selectable marker gene for targeted chromosomal integration during genetic manipulations of trypanosomatids.