An Efficient Method for Stable Transfection of Bloodstream-Form Trypanosoma Brucei

Since the original reports of electroporation of African trypanosomes ( 1,2), both transient and stable transfection of procyclic Trypanosoma brucei have now become routine. However, transfection of bloodstream-form parasites still remains technically difficult. Originally, bloodstream-form transfectants were obtained by first transfecting the procyclic form, then passing the procyclic form through the tsetse fly vector ( 3). Other techniques relied on parasite amplification in mammals, or involved the use of agarose plate cloning ( 4). The efficiency of these methods has remained generally low, and the results are somewhat unpredictable. Here we present a high efficiency method for stable transfection that involves no use of animals, nor of agarose plates. Our method is simple, direct and efficient. T.brucei brucei stock 427-60 bloodstream forms (variant 118a) were cultured in HMI-9 media ( 5) supplemented with 10% fetal calf serum (Gibco, Life Technology Inc., Grand Island, NY) and 10% Serum Plus (JRH Biosciences, Lenexa, KS) at 37 C and 5% C02. Overnight cultures that appeared healthy (usually at a density of 1–5 × 106 cells/ml) were used for transfection. Approximately 1–2 × 108 trypanosomes were collected, washed once in PBS at room temperature, centrifuged at 2500 r.p.m., 4 C for 8 min, and resuspended in 0.4 ml of ice cold ZPFMG ( 4) electroporation buffer (132 mM NaCl, 8 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, 0.5 mM magnesium acetate, 0.09 mM calcium acetate, 55 mM glucose, pH adjusted to 7.0 with acetic acid). Then, about 5–15 μg of linearized plasmid DNA (6) was mixed into the suspension, which was then rapidly transferred to a BTX 0.2 cm electroporation cuvette. Electroporation was immediately performed at a single pulse with a BTX 600 Electro Cell Manipulator (BTX Inc., San Diego, CA) at 1.0–1.2 kV, 25 μF and R6 resistance (186 Ω). Only ∼10% of the initial input cell population survived the electroporation process. After the electrical charge the cuvette containing the cell suspension was put on ice for an additional 10 min. Then the cell suspension was transferred into 20 ml HMI-9 medium with no selection drugs and mixed well. The diluted cell suspension was divided into five flasks (4 ml per flask) and put into an incubator. After 24 h of recovery, the cells were typically at a density of 0.5–1 × 106/ml. After counting, the cells were diluted to a concentration of 5 × 104 to 1 × 105 cells/ml with selection media, and 0.5 ml was transferred to each well of 24-well plates (Flow laboratories, Inc., McLean, Virginia). Typically, cells were selected in 0.5 μg/ml hygromycin-B (Boehringer Mannheim, Figure 1. Outline of the vector constructs and homologous recombination strategy for stable transfection of bloodstream-form T.brucei . The scheme shows the ODC gene locus and the plasmid constructs p31 and p409. In these antibiotic resistant gene cassettes the hygromycin-B-phosphotransferase (Hph) gene, or the phleomycin-resistance (Phleo) gene is flanked by the PARP splice acceptor site (s) and the βα-tubulin intergenic region polyadenylation sequences (i) (8). Targeting sequences as indicated are the 5 ′ 0.9 kb SacI–SacII fragment and the 3 ′ 2 kb SacII–SacI ODC locus genomic fragments (7). The arrows indicate the direction of an unidentified promoter driving the ODC gene and the antibiotic resistance genes after integration into the chromosome.