A retroviral expression system based on tetracycline-regulated tricistronic transactivator/repressor vectors for functional analyses of antiproliferative and toxic genes.
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.
Ausserlechner
Michael J
MJ
Molecular Biology Research Laboratory, Pediatric Department, Medical University Innsbruck, Innrain 66, A-6020 Innsbruck, Austria. michael.j.ausserlechner@uibk.ac.at
Obexer
Petra
P
Deutschmann
Andrea
A
Geiger
Kathrin
K
Kofler
Reinhard
R
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
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