Open AccessSIRNA-MEDIATED INHIBITION OF INTERLEUKINE-13 PRODUCTION IN VITRO
Author(s) -
I.P. Shilovskiy,
Dmitriy Mazurov,
Н Н Шершакова,
Musa Khaitov
Publication year - 2012
Publication title -
rossijskij allergologičeskij žurnal
Language(s) - English
Resource type - Journals
eISSN - 2686-682X
pISSN - 1810-8830
DOI - 10.36691/rja683
Subject(s) - lipofectamine , microbiology and biotechnology , transfection , in vitro , gene expression , messenger rna , small interfering rna , rna interference , complementary dna , gene , green fluorescent protein , biology , in vivo , chemistry , rna , vector (molecular biology) , recombinant dna , biochemistry , genetics
Background. According to current views, one of the major mediators involved in the development of allergic process is IL-13. The goal of this work was to design small interfering RNA molecules to effectively inhibit il-13 gene expression of mice in experiments in vitro. Methods. For the expression of IL-13 in in vitro gene coding sequence il-13 was amplified using cDNA ConA-stimulated spleen cells from BALE / c mice as a template and cloned into the expression vector pUCHR IRES GFP. Using a computer analysis were designed six variants of siRNA, directed against mRNA-il-13. To test the efficiency of siRNA a co-transfection of 1x 105 cells HEK293T mixture (0,5 mg and 1 mg of plasmid siRNA) coupled with Lipofectamine 2000 reagent was carried out. Twenty-four hours later, the gene expression changes in il-13 recorded by flow cytometry on the fluorescence intensity of GFP+-cells. Gene expression of il-13 mRNA was assessed by quantitative PCR, and the level of the protein product by ELISA. results. As a result, siRNA molecules were obtained and three of them were able to effectively inhibit the gene expression of il-13. Conclusion. Thereby variants of siRNA, which can effectively inhibit the production of mice’s IL-13 in vitro; can be used later in experiments in vivo so to understand the role of IL-13 in the pathogenesis of allergic conditions as to develop new therapy approaches.