In order to transfect T. evansi RoTat 1.2 parasites, it was necessary to define the conditions required for the successful integration of recombinant plasmids in trypanosomes when using the 4D-Nucleofector™ system (Lonza, Levallois-Perret, France). Following recommendations from the manufacturer, an optimization experiment that combines 23 different Nucleofector™ programs with 5 different solutions (P1–P5) for a total of 115 reactions was set up.
Briefly, for each reaction, a pellet of 106 parasites was resuspended in 20 µL of solutions P1–P5 along with 1 µg of NotI-linearized pLEW20-green fluorescent protein (GFP), a plasmid that was previously used for GFP expression in T. brucei (Wirtz et al. Reference Wirtz, Leal, Ochatt and Cross1999; Coustou et al. Reference Coustou, Guegan, Plazolles and Baltz2010), and transfected using one of the programs recommended by the manufacturer. Transfected parasites were first cultured for 24 h in the supplemented IMDM medium (Hirumi and Hirumi, Reference Hirumi and Hirumi1989) before addition of 2 µg mL-1 of phleomycin for the selection of viable clones. Successful clones were analysed for GFP expression using flow cytometry. The successful combination of transfection program and P1–P5 solutions from the Nucleofector™ kit was established for gene knock-out experiments.
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The matrix of 115 conditions recommended by the manufacturer yielded two combinations that produced viable clones: the program EF100 combined with solution P3 and the program DI100 with solution P5. Following the transfection, the potential recombinants were analysed for GFP fluorescence using flow cytometry (Supplementary Fig. S1). The combination of Program DI100 with solution P5 produced a higher number of clones when compared with program EF100 combined with solution P3 (results not shown) and was therefore chosen for future experiments.