An episomal vector for stable tetracycline-regulated gene expression.
The recently introduced tetracycline (Tc)-regulatable eukaryotic gene expression system based on the Escherichia coli Tn 10 tetracycline operon has proven to be a powerful tool for controlled expression of a variety of genes in vitro as well as in vivo . Control elements of this expression system are contained in two separate plasmid vectors. The tTA vector encodes a transactivator protein and the tetP vector contains a responsive operator-promoter element (tetP) that controls gene expression depending on tTA binding. Establishment of cell lines expressing a gene of interest under tetP control requires two subsequent rounds of transfection and clonal selection after each transfection. Here we describe a modification of this system in which the tetP element is placed in an episomal EBNA-based plasmid that can be stably maintained in primate but not in rodent cells. Using HeLa and human melanoma cells, we show that upon transient or stable transfection a reporter gene is expressed in a Tc-regulated manner similar to the original system. Thus, this expression system combines the advantages of episomal vectors, such as high efficiency of transfection and time-efficient selection of mass cultures, with tight control of gene expression provided by the Tc-regulatable system.
Jost
M
M
The Wistar Institute of Anatomy and Biology, 3601 Spruce Street, Philadelphia, PA 19104, USA.
Kari
C
C
Rodeck
U
U
D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics; D010957:Plasmids; D013752:Tetracycline / Q000494:pharmacology*; D014162:Transfection; D014407:Tumor Cells, Cultured
D008156:Luciferases; D013752:Tetracycline