Plasmid complementation of LdUMSBP in LdU−/+ and LdU−/− parasites

Plasmid complementation experiment was carried out as described earlier [30]. LdU^−/+ parasite was transfected with the pXG-PHLEO-LdUMSBP plasmid prepared by cloning LdUMSBP ORF in pXG-PHLEO in BamHI site (pXG-PHLEO vector was kind gift from Dr. Stephen M. Beverley, Department of Molecular Microbiology, Washington University in St. Louis) and selected with minimal doses of phleomycin (25 μg/mL) and finally grown in the presence of 50 μg/mL of drug. Also, LdU^−/− parasite was transfected with pXG-PHLEO-LdUMSBP plasmid and maintained initially with (25 μg/mL) of phleomycin and finally with 50 μg/mL. The selected cell pools were designated “LdU^−/+ AB” and “LdU^−/− AB” for UMSBP deleted, added back. The presence of episome in AB clones was determined by selectively amplifying LdUMSBP gene using specific pXG-PHLEO backbone primer (5′- CCTCCCCCTGTCCCCGGG-3′) and LdUMSBP reverse one.