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. Recent development of gene transfection systems in G. lamblia has allowed constitutive 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 with insertions of multiple tet operator sequences in the vicinity of a synthetic ran promoter were tested for their inducibility of a luciferase reporter gene expression. Stable cell lines transfected with individual plasmid constructs were established under drug selection. By assaying luciferase activity in transfected cells in response to tetracycline, an inducible promoter with insertion of two tet 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 further improve its inducibility, several other synthetic promoter contexts were also tested to increase expression of the tet-repressor gene. An optimal inducibility of 50-fold was obtained when a synthetic alpha-giardin promoter was used. Fine tuning of luciferase expression was achieved by adjusting the concentration of tetracycline and duration of drug exposure. The inducible 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 previously be achieved.
Sun
C H
CH
Division of Infectious Diseases, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, ROC.
Tai
J H
JH
D000818:Animals; D002460:Cell Line; D016054:DNA, Protozoan; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D016829:Giardia lamblia / Q000235:genetics* / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D009875:Operator Regions, Genetic; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics; D013752:Tetracycline / Q000494:pharmacology*; D014162:Transfection
D016054:DNA, Protozoan; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D008156:Luciferases; D013752:Tetracycline