A Comparison of Target gene Silencing using Synthetically Modified siRNA and shRNA That Express Recombinant Lentiviral Vectors

P V Spirin; Spirin P V; D Baskaran; Baskaran D; P M Rubtsov; Rubtsov P M; M A Zenkova; Zenkova M A; V V Vlassov; Vlassov V V; E L Chernolovskaya; Chernolovskaya E L; V S Prassolov; Prassolov V S

doi:10.32607/20758251-2009-1-2-86-90

A Comparison of Target gene Silencing using Synthetically Modified siRNA and shRNA That Express Recombinant Lentiviral Vectors

Authors: Spirin PV¹^,2, Baskaran D¹^,2, Rubtsov PM¹^,2, Zenkova MA¹^,2, Vlassov VV¹^,2, Chernolovskaya EL¹^,2, Prassolov VS¹^,2
Affiliations:
1. Engelghardt Institute of Molecular Biology, Russian Academy of Sciences
2. Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences
Issue: Vol 1, No 2 (2009)
Pages: 86-90
Section: Articles
URL: http://actanaturae.ru/2075-8251/article/view/10819
DOI: https://doi.org/10.32607/20758251-2009-1-2-86-90
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Abstract

RN A interference is an efficient natural mechanism of gene expression modulation on the post translation level that was revealed both in higher animal and plant eukaryotes and in lower level eukaryotes and viruses. At present RN A interference is used as a powerful instrument in research of the functional activity of the genes, and with the help of this instrument findings have been obtained that are of significant importance for many areas of fundamental biology. At the same time, developments are under way in many world laboratories aimed at creating new-generation therapeutic means able to treat inherited, malignant and inflectional diseases of different aetiology based on the usage of the interfering RN As. One of the major problems of these researches is the retrieval of the efficient techniques able to deliver the interfering RN As into the target cells. At present for the introduction of the interfering RN As into the cells transfection and transduction are used with the help of the viral vectors that direct the shRN A synthesis in the cells, which are the predecessors of the corresponding siRN As. In the article findings are given of the comparison the efficiency of the oncogene AML1-ET O suppression with the help of lipofection of the synthetic siRN A and also while using the lentiviral vector which directs the shRN A synthesis – the anti AML1- ET O siРНК predecessor.

Keywords

retroviral vectors, RN A interference, embryonic mouse fibroblasts.

The controlled silencing of target genes is important both for molecular biological studies and for related applied sciences: in particular, modern biomedicine. Among the many gene silencing approaches (which include the use of anti-sense RN A, ribozymes, chemical blockers, and targeted mutagenesis), the most efficient approach is based on RN A-interference. RN A interference is a highly specific mechanism for the posttranscriptional silencing of target genes. It involves the degradation of the target gene mRN A. The degradation of mRN A occurs in a complex formed by short-interfering RN A oligonucleotides (siRN A) and cellular proteins such as endonucleases. The nucleotide sequence of siRN A is complementary to that of target gene mRN A. In the past couple of years, the use of siRN A has become widespread in studies of gene functioning and gene interaction. The use of siRN A as next generation therapeutic agents in biomedicine is also being explored. It is possible that, in the near future, siRN A will be used for treating viral and oncological diseases. Currently, short synthetic 21–22-bp double-stranded siRNA molecules are widely used to silence mammalian genes. A number of commercial firms synthesize siRN A oligonucleotides. These commercial firms have siRN A design tools available on their websites (e.g., www.qiagen.com). Synthetic siRN A oligonucleotides are transferred into cells in vitro by lipofection. Since siRN A induces the degradation of mRN A (and not the protein directly), the silencing effect does not occur immediately after cell transfection. The silencing effect is generally noticeable within 18 hours of transfection: however, in the case of stable proteins, the silencing effect may be noticeable only 24–48 hours after transfection. The longevity of siRN A silencing is comparatively short, and different sources claim that the silencing effect lasts for 3–5 cell divisions. It should be noted that the longevity of siRN A silencing may depend on many factors, in particular the nature of the cells being transfected. Approaches have been developed to synthetically modify siRN A oligonucleotides, which enhance the longevity of siRN A silencing in cells. Such synthetically modified siRN A oligonucleotides are useful for the post-transcriptional silencing of genes that encode proteins with a long half-life.