title: Fast set-up of doxycycline-inducible protein expression in human cell lines with a single plasmid based on Epstein-Barr virus replication and the simple tetracycline repressor.
abstract: We have developed a novel plasmid vector, pEBTetD, for full establishment of doxycycline-inducible protein expression by just a single transfection. pEBTetD contains an Epstein-Barr virus origin of replication for stable and efficient episomal propagation in human cell lines, a cassette for continuous expression of the simple tetracycline repressor, and a cytomegalovirus-type 2 tetracycline operator (tetO2)-tetO2 promoter. As there is no integration of vector into the genome, clonal isolation of transfected cells is not necessary. Cells are thus ready for use 1 week after transfection; this contrasts with 3-12 weeks for other systems. Adequate regulation of protein expression was accomplished by abrogation of mRNA polyadenylation. In northern analysis of seven cDNAs coding for transport proteins, pools of transfected human embryonic kidney 293 cells showed on/off mRNA ratios in the order of 100:1. Cell pools were also analyzed for regulation of protein function. With two transport proteins of the plasma membrane, the on/off activity ratios were 24:1 and 34:1, respectively. With enhanced green fluorescent protein, a 23:1 ratio was observed based on fluorescence intensity data from flow cytometry. The unique advantage of our system rests on the unmodified tetracycline repressor, which is less likely, by relocation upon binding of doxycycline, to cause cellular disturbances than chimera of tetracycline repressor and eukaryotic transactivation domains. Thus, in a comprehensive comparison of on- and off-states, a steady cellular background is provided. Finally, in contrast to a system based on Flp recombinase, the set-up of our system is inherently reliable.
authors.affiliation: Department of Pharmacology, University of Cologne, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Bach
authors.forename: Silke
authors.initials: S
authors.lastname: Grigat
authors.forename: Barbara
authors.initials: B
authors.lastname: Pawlik
authors.forename: Christian
authors.initials: C
authors.lastname: Fork
authors.forename: Olaf
authors.initials: O
authors.lastname: Utermöhlen
authors.forename: Sonia
authors.initials: S
authors.lastname: Pal
authors.forename: David
authors.initials: D
authors.lastname: Banczyk
authors.forename: Andreas
authors.initials: A
authors.lastname: Lazar
authors.forename: Edgar
authors.initials: E
authors.lastname: Schömig
authors.forename: Dirk
authors.initials: D
authors.lastname: Gründemann
mesh_terms: D015152:Blotting, Northern; D002460:Cell Line; D004318:Doxycycline / Q000494:pharmacology*; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D010957:Plasmids / Q000235:genetics*; D014176:Protein Biosynthesis / Q000187:drug effects; D018741:Replication Origin / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D013752:Tetracycline / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D014779:Virus Replication / Q000235:genetics
chemical_list: D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D013752:Tetracycline; D004318:Doxycycline