Fig 2. Optimization of pLEXSY-iRFP vector by introducing downstream regions (DSR) of different genes.

A) Schematic view of pLEXSY-hyg2 constructs containing infrared (iRFP) reporter gene and different DSR corresponding to A2, AMASTIN and HSP70-II genes. Constructs were linearized by SwaI digestion prior to electroporation. B) Correlation between fluorescence signal and the number of logarithmic promastigotes modified with iRFP-pLEXSY-HYG (□) or carrying the DSR of A2 (●), AMASTIN (○) or HSP70-II (Δ) genes. C) Flow-cytometry analysis of intracellular amastigotes isolated from THP-1 in vitro infections. D) Mean fluorescence intensity emitted by lesion-derived amastigotes obtained from WT and the engineered parasite strains carrying iRFP. Experiments were carried out with 10⁷ parasites by triplicate and error bars represent standard deviations (SD). Significance level *** p< 0,001; ** p<0.01; from two tails of Student t-test.