F Olmo1; C Rotger2; M C Taylor1; F Orvay2; A Costa2; J M Kelly1;
1 London School of Hygiene and Tropical Medicine, UK; 2 Universitat de les Illes Balears, Spain
DiscussionElectroporation is the only technique routinely used to transfect trypanosomatids.
However, the procedure has limited efficiency and flexibility, for example, taking
up to 6 weeks to establish a genetically transformed culture of Trypanosoma cruzi. We have developed a new method of transfection, using a non-viral, toxicity-free chemical carrier, which is able to efficiently package DNA into globular particles (visible under atomic force microscopy). The DNA-carrier complex is efficiently internalized without damaging the parasite or disrupting the cell cycle. The procedure involves a very straightforward 3-step protocol, without the need for expensive devices, reagents and cuvettes, and can generate >3000 stable transformants per microgram of plasmid DNA. The approach has been applied to several intra and extra-cellular trypanosomatid species using both episomal and integrative vectors. Parasites expressing RFP/GFP are visible within 6 hours and they can be cultured under drug selection to generate stably transformed lines. This approach has great potential to help address new and important biomedical questions, related to pathogenesis, drug-resistance and parasite latency. Recently obtained results also suggest that the technique will be more widely applicable to other pathogens.