Development of a tetracycline controlled gene expression system in the parasitic protozoan Giardia lamblia

Giardia lamblia is a very common intestinal protozoan pathogen of humans. R ecent development of gene transfection systems in G. lamblia has allowed co nstitutive expression of selected genes in the organism. To extend the uses of DNA transfection in G. lamblia an inducible gene expression system was developed by integrating the bacterial tet operator-repressor elements into an episomal DNA transfection vector. Tetracycline-responsive promoters wit h insertions of multiple rer operator sequences in the vicinity of a synthe tic ran promoter were tested for their inducibility of a luciferase reporte r gene expression. Stable cell lines transfected with individual plasmid co nstructs were established under drug selection. By assaying luciferase acti vity in transfected cells in response to tetracycline, an inducible promote r with insertion of two let operators downstream of the adjacent synthetic ran promoter was found to confer a 10-fold inducibility in gene expression with co-expression of the tet-repressor driven by a gdh promoter. To furthe r improve its inducibility, several other synthetic promoter contexts were also tested to increase expression of the tel-repressor gene. An optimal in ducibility of 50-fold was obtained when a synthetic alpha-giardin promoter was used. Fine tuning of luciferase expression was achieved by adjusting th e concentration of tetracycline and duration of drug exposure. The inducibl e gene expression system provides us an easy way to manipulate the level of gene expression in G. lamblia in a controllable manner that could not prev iously be achieved. (C) 2000 Elsevier Science B.V. All rights reserved.