Overexpressing Leishmania ProstaglandinF2S

The LbrPGF2S CDS (coding DNA sequence) was amplified from the genomic DNA (gDNA) of L. (V.) braziliensis strain MHOM/BR/75/M2904 using primers LbrPGF2S-nostart-BglII-For (5ʹ-TCA AGA TCT GCT GGG GCC GCT GGG GCC ATC AAC GTT GGT AAG ACC G -3ʹ) and LbrPGF2S-BamHI-Rev (5ʹ-TCA GGA TCC TCA GAA CTG CGC CTC ATC A -3ʹ). The PCR products were digested with BglII and BamHI enzymes and cloned into the pmCherry-C1 (Addgene, Cambridge, MA, USA) plasmid digested with same enzymes. The mCherry-PGF2S plasmid was then digested with PmeI and NdeI enzymes. Promastigotes were transfected with mCherry-PGFS or mCherry linear fragments by electroporation [16 (link)]. Transfectant colonies were extracted from M199-agar medium in the presence of the G418 antibiotic (Sigma-Aldrich, St. Louis, MO, USA). The G418 LD₅₀ was determined for the Lb2903 strain, and at four-fold LD₅₀ drug concentration (4 µg/ml). Parasites overexpressing LbrPGF2S ectopically were produced and kept as described in [4 (link)].