title: A retroviral expression system based on tetracycline-regulated tricistronic transactivator/repressor vectors for functional analyses of antiproliferative and toxic genes.
abstract: Establishment of stably transfected mammalian cells with conditional expression of antiproliferative or proapoptotic proteins is often hampered by varying expression within bulk-selected cells and high background in the absence of the inducing drug. To overcome such limitations, we designed a gene expression system that transcribes the tetracycline-dependent rtTA2-M2-activator, TRSID-silencer, and selection marker as a tricistronic mRNA from a single retroviral vector. More than 92% of bulk-selected cells expressed enhanced green fluorescent protein or luciferase over more than three orders of magnitude in an almost linear, dose-dependent manner. To functionally test this system, we studied how dose-dependent expression of p27(Kip1) affects proliferation and viability of SH-EP neuroblastoma cells. Low to moderate p27(Kip1) expression caused transient G(0)-G(1) accumulation without reduced viability, whereas high p27(Kip1) levels induced significant apoptosis after 72 hours. This proves that this expression system allows concentration-dependent analysis of gene function and implicates p27(Kip1) as a critical regulator of both proliferation and apoptosis in SH-EP neuroblastoma cells.
authors.affiliation: Molecular Biology Research Laboratory, Pediatric Department, Medical University Innsbruck, Innrain 66, A-6020 Innsbruck, Austria. michael.j.ausserlechner@uibk.ac.at
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Ausserlechner
authors.forename: Petra
authors.initials: P
authors.lastname: Obexer
authors.forename: Andrea
authors.initials: A
authors.lastname: Deutschmann
authors.forename: Kathrin
authors.initials: K
authors.lastname: Geiger
authors.forename: Reinhard
authors.initials: R
authors.lastname: Kofler
mesh_terms: D017209:Apoptosis / Q000235:genetics*; D002453:Cell Cycle / Q000235:genetics; D049109:Cell Proliferation; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004318:Doxycycline / Q000494:pharmacology; D020869:Gene Expression Profiling / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D009447:Neuroblastoma / Q000235:genetics / Q000473:pathology; D012097:Repressor Proteins / Q000187:drug effects / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics*; D013752:Tetracycline / Q000494:pharmacology*; D015534:Trans-Activators / Q000187:drug effects / Q000235:genetics*; D014162:Transfection; D051816:Tristetraprolin / Q000187:drug effects / Q000235:genetics; D014407:Tumor Cells, Cultured
chemical_list: C436291:CDKN1B protein, human; D047908:Intracellular Signaling Peptides and Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D051816:Tristetraprolin; D049452:Green Fluorescent Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D013752:Tetracycline; D004318:Doxycycline