Development of Leishmania Species Strains with Constitutive Expression of eGFP

Protozoan parasites of the genus Leishmania cause leishmaniasis, a disease with variable clinical manifestations that affects millions of people worldwide. Infection with L. donovani can result in fatal visceral disease. In Panama, Colombia, and Costa Rica, L. panamensis is responsible for most of the reported cases of cutaneous and mucocutaneous leishmaniasis. Studying a large number of drug candidates with the methodologies available to date is quite difficult, given that they are very laborious for evaluating the activity of compounds against intracellular forms of the parasite or for performing in vivo assays. In this work, we describe the generation of L. panamensis and L. donovani strains with constitutive expression of the gene that encodes for an enhanced green fluorescent protein (eGFP) integrated into the locus that encodes for 18S rRNA (ssu). The gene encoding eGFP was obtained from a commercial vector and amplified by polymerase chain reaction (PCR) to enrich it and add restriction sites for the BglII and KpnI enzymes. The eGFP amplicon was isolated by agarose gel purification, digested with the enzymes BglII and KpnI, and ligated into the Leishmania expression vector pLEXSY-sat2.1 previously digested with the same set of enzymes. The expression vector with the cloned gene was propagated in E. coli, purified, and the presence of the insert was verified by colony PCR. The purified plasmid was linearized and used to transfect L. donovani and L. panamensis parasites. The integration of the gene was verified by PCR. The expression of the eGFP gene was evaluated by flow cytometry. Fluorescent parasites were cloned by limiting dilution, and clones with the highest fluorescence intensity were selected using flow cytometry.