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 Trypanosoma brucei to the nucleoside antibiotic blasticidin S were assessed. A concentration of 10 microg 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 microg 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.
Brooks
D R
DR
Wellcome Centre for Molecular Parasitology, University of Glasgow, The Anderson College, Glasgow, UK. d.brooks@udcf.gla.ac.uk
McCulloch
R
R
Coombs
G H
GH
Mottram
J C
JC
D000619:Aminohydrolases / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D030801:Animals, Genetically Modified; D003546:Cysteine Endopeptidases / Q000235:genetics*; D005819:Genetic Markers; D007894:Leishmania mexicana / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D016254:Mutagenesis, Insertional; D009705:Nucleosides / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014170:Transformation, Genetic; D014344:Trypanocidal Agents / Q000494:pharmacology*; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*
D005819:Genetic Markers; D009705:Nucleosides; D011994:Recombinant Proteins; D014344:Trypanocidal Agents; C004500:blasticidin S; D003546:Cysteine Endopeptidases; D000619:Aminohydrolases; C019795:blasticidin S deaminase