Up-regulation of luciferase gene expression with antisense oligonucleotides: implications and applications in functional assay development.
HeLa Tet-Off cells were transfected transiently as well as stably with a recombinant plasmid (pLuc/705) carrying the luciferase gene interrupted by a mutated human beta-globin intron 2 (IVS2-705). The mutation in the intron causes aberrant splicing of luciferase pre-mRNA, preventing translation of luciferase. However, treatment of the cells with a 2'-O-methyl-oligoribonucleotide targeted to the aberrant splice sites induces correct splicing, restoring luciferase activity. The effects are sequence-specific, depend on the concentration of the oligonucleotide, and can be modulated by the pretreatment of the cell line, Luc/705, with tetracycline. Thus, the cell line provides, among others, a novel functional assay system superior to other procedures that are based on protein down-regulation. In particular, the system would be ideal in assessing the cellular delivery efficiency of antisense oligonucleotides.
Kang
S H
SH
School of Pharmacy, Department of Pharmacology, and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Cho
M J
MJ
Kole
R
R
D017398:Alternative Splicing; D002460:Cell Line; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D017354:Point Mutation; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012322:RNA Precursors / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology; D014162:Transfection; D015854:Up-Regulation
D016376:Oligonucleotides, Antisense; D011500:Protein Synthesis Inhibitors; D012322:RNA Precursors; D008156:Luciferases; D013752:Tetracycline