Production of Recombinant Leishmania Tropica Parasites Expressing Two Proteins of Egfp and Luciferase Reporter

Generation of recombinant Leishmania tropica (L. tropica) expressing two reporter proteins EGFP and LUC L. tropica is the major cause of cutaneous and visceral leishmaniasis in Iran. Due to lack of an accurate, sensitive and noninvasive detection test in animal model is a major problem. Here, we designed L. tropica stably transfected with two reporter genes as fused form, enhanced green fluorescent protein (egfp) and Luciferase (luc), named as egfp-luc to use as a specific tools for detection and measurement of parasite load in live animals.
In an experimental study, linearized cassette containing egfp-luc genes was stably integrated into Iranian strain of wild-type L. tropica genome, 18srRNA locus, by homologous recombination. Transfectants were screened by G418 resistance, confirmed by PCR and western blotting and the expression of EGFP signals were observed by fluorescent microscope and flow cytometry.
FINDINGS: Phenotype observations (by fluorescent microscopy and flow cytometry) were shown that recombinant L. tropicaEGFP-LUC was successfully produced EGFP protein into cytoplasm. Quantification of EGFP intensity was more than 90%. Furthermore, the results of PCR and western blotting verified the proper integration into genome and expression of both genes in promastigotes.
This is the first report to show the generation of recombinant L. tropicaEGFP-LUC expressing two reporter genes simultaneously.