Thebleresistance gene as a new selectable marker forTrypanosoma brucei: fly transmission of stable procyclic transformants to produce antibiotic resistant bloodstream forms

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Abstract
We describe here the stable transformation of Trypanosoma brucei using a new selectable marker for klnetoplastld protozoa, the Sh ble, or phleomycln, resistance gene. A plasmld containing this gene targeted to the tubulln gene locus by homologous sequences was introduced Into procyclic trypanosomes by electroporation and cells selected for antibiotic resistance. Southern analysis of stable transformants showed that the plasmld had been Integrated Into the tubulln locus by homologous recombination. Analysis of bloodstream stage transformants, produced by transmission through the vector Glossina, showed that the resistance gene was conserved and expressed In these forms in the absence of selective drug pressure. In both procyclic and bloodstream forms, transcription of the ble gene appears to originate from the upstream tubulln promoter, despite the presence of a VSG promoter In the integrated construct. The generation of stable bloodstream transformants for the first time will facilitate the study of gene function and expression during the trypanosome life cycle, and aid in the investigation of genetic exchange in these organisms.