TAILIEUCHUNG - Báo cáo Y học: Selection of effective antisense oligodeoxynucleotides with a green fluorescent protein-based assay Discovery of selective and potent inhibitors of glutathione S-transferase Mu expression

Antisense oligodeoxynucleotides (AS-ODNs) are frequently used for the down-regulation of protein the majority of potential antisense sequences lacks effect-iveness, fast screening methods for the selection of effective AS-ODNs are describe a new cellular screening assay for the evaluationof the potency and specificityof new antisense sequences. | Eur. J. Biochem. 269 2574-2583 2002 FEBS 2002 doi Selection of effective antisense oligodeoxynucleotides with a green fluorescent protein-based assay Discovery of selective and potent inhibitors of glutathione S-transferase Mu expression Peter A. C. t Hoen1 2 Bram-Sieben Rosema1 Jan N. M. Commandeur2 Nico P. E. Vermeulen2 Muthiah Manoharan3 Theo J. C. van Berkel1 Eric A. L. Biessen1 and Martin K. Bijsterbosch1 1 Division of Biopharmaceutics LeidenjAmsterdam Center for Drug Research the Netherlands 2Division of Molecular Toxicology LeidenjAmsterdam Center for Drug Research the Netherlands 3 ISIS Pharmaceuticals Carlsbad California USA Antisense oligodeoxynucleotides AS-ODNs are frequently used for the down-regulation of protein expression. Because the majority of potential antisense sequences lacks effectiveness fast screening methods for the selection of effective AS-ODNs are needed. We cesscrb e a new haUular screening assay for the evaluation of the potency and specificity of new antisense sequences. Fusion constructs of ithe gene ol i n eerest and the gene encoding the enhanced green fluorescent protein EGFP are cotransfected with AS-ODNs to COS-7 cells. Subsequently cells are analyeed for expression of the EGFP fusion protein by flow cytometry. With the assay we tested the effectiveness of a set of 15 phosphorothioate ODNs against rat glutathione S-transferase Mu1 GSTM1 and or Mu2 GSTM2 . We fiuund eeveral AS-ODNs that demonstrated potent sequence-specific and concentrationdependent inhibition of fusion protein M AS-6 and AS-8 inhibited EGFP-GSTM1 expression by 95 4 and81 6 respectively. AS-5 and AS-10 were selective for GSTM2 82 4 and 85 decrease respectively . AS-2 ndd AS-3 laitteted at liomolgouus regions in GSTM1 and GSTM2 inhibited both isoforms 77-95 decrease . Oflrrr AS-ODNs were not etVeaiire or displayed non-target-specific inhibition of protein expression. The observed decrease m EGH eppi oon WHS

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