A retroviral expression system based on tetracycline-regulated tricistronic transactivator/repressor vectors for functional analyses of antiproliferative and toxic genes
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E-Artikel
Titel:
A retroviral expression system based on tetracycline-regulated tricistronic transactivator/repressor vectors for functional analyses of antiproliferative and toxic genes
Beschreibung:
<jats:title>Abstract</jats:title>
<jats:p>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 p27Kip1 affects proliferation and viability of SH-EP neuroblastoma cells. Low to moderate p27Kip1 expression caused transient G0-G1 accumulation without reduced viability, whereas high p27Kip1 levels induced significant apoptosis after 72 hours. This proves that this expression system allows concentration-dependent analysis of gene function and implicates p27Kip1 as a critical regulator of both proliferation and apoptosis in SH-EP neuroblastoma cells. [Mol Cancer Ther 2006;5(8):1927–34]</jats:p>