A tetracycline-inducible gene expression system in Entamoeba histolytica.
We have developed an episomal inducible gene expression system in Entamoeba histolytica based on the TetR repressor. The tetR gene was placed under control of 5' and 3' ferredoxin (fdx) regulatory sequences on a plasmid encoding the hygromycin resistance gene directed by 5' and 3' hgl sequences. The reporter luciferase constructs were introduced on a second episome bearing the neomycin resistance gene controlled by 5' and 3' actin sequences. The reporter constructs were driven by the hgl5 promoter in which the tetO sequence was introduced. We found that the optimal tetO location for induction by tetracycline was +4 from the start of transcription. The efficiency of repression and the induction ratio could be improved by increasing hygromycin levels, presumably by increasing tetR plasmid levels. Under these conditions, maximal induction of reporter luciferase could be effected with 5 micrograms/ml tetracycline in 18 h. This system permits regulated expression of the reporter gene over two orders of magnitude and should be useful in the analysis of gene function.
Ramakrishnan
G
G
Department of Medicine, University of Virginia Health Sciences Centre, Charlottesville 22908, USA.
Vines
R R
RR
Mann
B J
BJ
Petri
W A
WA
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002934:Cinnamates; D004351:Drug Resistance / Q000235:genetics; D004748:Entamoeba histolytica / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects; D017125:Genes, Protozoan / Q000235:genetics*; D017930:Genes, Reporter; D005822:Genetic Vectors; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics*; D013752:Tetracycline; D014162:Transfection
D000900:Anti-Bacterial Agents; D002934:Cinnamates; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D006921:Hygromycin B; C026273:hygromycin A; D008156:Luciferases; D013752:Tetracycline