Colony polymerase chain reaction of stably transfected Trypanosoma cruzi grown on solid medium

Tools for the genetic manipulation of Trypanosoma cruzi are largely, unavai lable, although several vectors for transfection of epimastigotes and expre ssion of foreign or recombinant genes have been developed. We have previous ly constructed several plasmid vectors in which recombinant genes are expre ssed in T. cruzi using the rRNA promoter. In this report, we demonstrate th at one of these vectors can simultaneously mediate expression of neomycin p hosphotransferase and green fluorescent protein when used to stably transfe ct cultured epimastigotes. These stably transfected epimastigotes can be se lected and clotted as unique colonies on solid medium. We describe a simple colony PCR approach to the screening of these T. cruzi colonies for releva nt genes. Thus, the methodologies outlined herein provide important new too ls for the genetic dissection of this important parasite.