Stable DNA transformation of Toxoplasma gondii using phleomycin selection

Toxoplasma gondii (Tg) is an obligate intracellular protozoan parasite that is an important opportunistic pathogen in humans. To provide additional tools for molecular genetic analysis, we have developed a set of vectors for DNA transformation in Tg based on selection with the antibiotic phleomycin (Ph). These new vectors rely on the flanking sequences from the parasite genes GRA1, GRA2 or SAG1 to drive expression of the Tn5 ble gene encoding resistance to the DNA intercalating drug Ph (PhR). Treatment of extracellular parasites was used to select stable PhR transformants by plaque formation on host cell monolayers. Transfection of linear or circular forms of the pGRA1/ble, pGRA2/ble or pSAG1/ble vectors by electroporation resulted in stable transformation with an efficiency of approx. 10(-4)/micrograms DNA. Stable transformants contained 1-5 copies of ble that were integrated at non-homologous sites in the parasite nuclear genome. Ble provides a new dominant selectable marker for safe, efficient and rapid isolation of stable DNA transformants in Tg.