CLONING OF SHRNA SEQUENCE SPECIFIC TO ELASTASE OF MACROPHAGES INTO EXPRESSION VECTOR PGPV-17019250
Abstract
BACKGROUND: Macrophage elastase/MMP12 is one of the most important matrix metalloproteinase that cells secrete into the extracellular space. Secreted and activated MMP12 is capable to digest the proteins of extracellular matrix, such as fibronectin, laminin and elastin, facilitating the migration of immune cells to the inflamed tissue.
AIM: The aim of our study was to create a vector that would express a specific small interfering RNA (shRNA) to suppress the expression of human macrophage elastase.
METHODS: The online tool "siRNA Wizard" was used to design cDNA encoding shRNA specific to human macrophage elastase. The restriction endonucleases BamH1 and EcoRI, as well as a commercial T4 DNA ligase we used to clone the named cDNA into the expression vector pGPV-17019250. The presence of the cDNA in the vector was confirmed by PCR and DNA-sequencing with vector-specific primers.
RESULTS: In this study, we obtained the sequence of cDNA, encoding shRNA specific to macrophage elastase. We also cloned the named cDNA into the expression vector pGPV-17019250 and confirmed its exact location in the vector.
CONCLUSION: The generated expression vector pGPV-17019250-EM designated to suppress human macrophage elastase in cultured cells.
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