Highly efficient stable transformation of bloodstream forms of Trypanosoma brucei.

With the completion of the sequence of the megabase hromosomes of Trypanosoma brucei [1], it has become traightforward to design primers and amplify the appropriate anking sequences to knock out genes by homologous recomination [2]. In addition, RNA interference can be used to own-regulate individual genes or gene families [3], and has lso been applied to genome-wide screens [4]. Despite these dvances in technology, genome-wide screens have yet to be pplied to some of the most important issues in trypanosome iology. These include antigenic variation, the phenomenon of uman serum-resistance by T. b. gambiense and T. b. rhodeiense, and the acquisition of drug-resistance, all of which need o be studied in bloodstream forms of the parasite. Unfortunately, t has not been possible to produce representative RNA interfernce libraries, or to use libraries for complementation, because f the extremely low rate of stable transformation in this stage f the life cycle. Stable transformation of trypanosomes with foreign DNA sually occurs by electroporation with a short high-voltage ulse followed by antibiotic selection. For procyclic forms, this ethod can give efficiencies of 10−3–10−6 [2,5]. In contrast, loodstream forms are considerably more difficult to transfect, s the survival rate of the cells is lower and, somewhat paraoxically, homologous recombination seems to be less efficient. here have been attempts to improve the transfection efficiency n bloodstream forms by using different culture conditions, uffers and electroporation parameters [6,7]. Nevertheless, the fficiency of stable transformation (10−7 to 10−8) is several rders of magnitudes lower than with procyclic forms. The appli-