Research Article: A Comparison of Target Gene Silencing using Synthetically Modified siRNA and shRNA That Express Recombinant Lentiviral Vectors

Date Published: July , 2009

Publisher: A.I. Gordeyev

Author(s): P.V. Spirin, D. Baskaran, P.M. Rubtsov, M.A. Zenkova, V.V. Vlassov, E.L. Chernolovskaya, V.S. Prassolov.

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Abstract

RNA-interference is an effective natural mechanism of post-transcriptional modulation of gene expression. RNA-interference mechanism exist as in high eukaryotes both animals and plants as well in lower eukaryotes and viruses. RNA-interference is now used as a powerful tool in study of functional gene activity and many essential for fundamental biology results was obtained with this approach. Also it’s widely believed that RNA-interference could be used in working out of new therapeutic medicine against malignant, infectious and hereditary diseases. One of the main problems of these developments is search of effective methods of siRNA transfer into the target cells. At present time for these purpose different sorts of transfect ions or viral transduction are used. At present article the results of comparison of inhibition of expression of oncogene AML-ET O by synthetic siRNA and by recombinant lentiviruses coding for corresponding shRNA are presented.

Partial Text

The controlled silencing of target genes is important both for molecular biological studies and for related applied sciences: in particular, modern biomedicine.

Our results illustrate the high efficiency of stable synthetically modified siRNA duplexes for silencing the activated oncogenes frequently found in acute myeloid leukemia patients. Validated siRNA sequences were used to design and synthesize shRNA-coding DNA sequences. The synthesized DNA sequences were cloned into a recombinant lentiviral vector. The shRNA-expressing lentiviral vector particles targeting the junction point of AML1-ETO mRNA and 5`-end of RUNX1 mRNA were used to transduce the oncogene-expressing model cell lines. The transduced model cell lines were analyzed by RT-PCR and flow cytometry. The analysis revealed a significant reduction in the expression of activated oncogenes in the transduced cell lines. This is indicative of the high efficiency of the constructed lentiviral vector constructs, which can be used for silencing target genes by RNA interference.

We would like to thank Prof. P.M. Chumakov for providing us with the pLSLP lentiviral vector. The experimental work was carried out within the framework of the “Molecular and Cell Biology” project and project No. 27 “Fundamental Research Basics in Nanotechnology and Nanomaterials” of the RAS presidium fundamental research program.

 

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