Jonathan H. Lebowitz

Simultaneous transient expression assays of the trypanosomatid parasite Leishmania using β-galactosidase and β-glucuronidase as reporter enzymes

Abstract We describe a transient transfection protocol for cultured Leishmania major promastigotes, utilizing Escherichia coli genes encoding β-galactosidase and β-glucuronidase inserted into an expression vector derived from the dihydrofolate reductase-thymidylate synthase locus. Less than 0.1 pg of either reporter enzyme can be detected with a simple fluorimetric assay, and transfection of 10 μg of either reporter construct yields activities at least 100-fold over background. Simultaneous introduction of both constructs showed that the activity of each reporter gene was unaffected by the presence of the other, allowing one reporter construct to serve as a control for experimental variability in test gene constructs containing the second reporter gene. These results show that it is feasible to apply transient expression assays to the identification of cis-acting elements of genes encoding nonabundant mRNAs in the genus Leishmania.

thymidine kinase as a negative selectable marker in Leishmania major

We have tested whether the thymidine kinase gene of herpes simplex virus 1 (HSV-1 tk) could function as a negative selectable marker in Leishmania major. Several drug resistance markers have been used for positive selection of transfected DNA in trypanosomatids, including neomycin phosphotransferase (NEO; refs. 1-4) and hygromycin phosphotransferase (HYG; refs. 5,6). Negative selectable markers, that is genes whose expression is lethal to cells, have also proven useful for a variety of purposes in prokaryotic and eukaryotic systems. For example, negative selections have been used to enrich for homologous versus random recombinants in cultured mammalian cells [7], to ablate specific cell lineages in transgenic mice after fusion of the negative marker to a cell type-specific enhancer/promoter [8-11], and to select for muta-