Stable transformation of pleomorphic bloodstream form Trypanosoma brucei

Abstract

African trypanosomes differentiate between various developmental stages both in mammalian hosts and their tsetse vector to adapt to and survive in the different environments they encounter. In the bloodstream, trypanosomes naturally exist as either proliferative slender-forms or non-proliferative stumpy-forms, the latter being responsible for both prolonged infection and transmission. However, most trypanosome studies are carried out on laboratory-adapted monomorphic cell lines, incapable of differentiating to stumpy-forms or completing the life cycle through the tsetse fly. Partly, this has been due to the inefficiency of transfection of pleomorphic strains which have retained the ability to generate stumpy-forms. Recently, Amaxa Nucleofector® technology was shown to increase transfection efficiency for monomorphic bloodstream forms. Using this technology we have optimised a similar method for pleomorphic bloodstream form transfection, generating transfection efficiencies of 10(-7)-10(-6). This permits routine genetic manipulation of pleomorphic lines, which have the most biological relevance for trypanosomes in the field.

Keywords: Pleomorphic; Stumpy; Transfection; Trypanosoma brucei; Trypanosome.

Fig. 1

Transfection of pleomorphic transfections from cells harvested from blood or from culture. (A) Diagram outlining the method for the harvest and transfection of pleomorphic slender forms (details described in main text). (B) Pleomorphic bloodstream form AnTat1.1 90:13 cells were harvested from mouse blood 3 days post infection or after 7 days in vitro. Between 2.8 and 4.06 × 10⁷ cells were transfected with 10 μg of pALC14 vector in the “Amaxa basic parasite nucleofector solution 2” transfection buffer and selected in 24 well plates at 1:2, 1:5, 1:25 and 1:125 dilutions with 0.5 μg/ml puromycin. Transfection efficiencies were calculated from the number of positive wells per dilution (excluding any dilution where >50% of wells were positive) and extrapolating for the total number of cells transfected. Replicates were carried out from three independent mouse infections and three independent in vitro cultures.