Production of Lentiviral Vector Expressing MicroRNA-148b

Abstract

Background: Micro (mi)RNAs are non-coding endogenous RNAs which regulate gene expression by hybridization to specific binding sites in target mRNA sequences. Since several miRNAs are involved in proliferation and differentiation, miRNA-based therapies could be promising approach in regenerative medicine. Among different vehicles, lentiviral vector system is suitable for miRNA delivery. Besides, it is shown that miRNA-148b is involved in osteogenic differentiation. In this study, designing and cloning of miR-148b to lentiviral vector were investigated. Methods: We introduced miRNA-148b-3p/-5p into lentiviral vector through cloning producers. The sequences of lentiviral vectors carrying miRNA-148b were checked via analytical digestion as well as Sanger DNA sequencing. In the following, produced lentiviral vectors were used for mesenchymal stem cells transduction. Findings: Designed miR-148b-3p/-5p successfully cloned to the shuttle. Correctness and absence of any unintended mutations of lentiviral shuttle carrying miRNA-148b3p/-5p were confirmed followed by lentiviral production. Expression of enhanced green fluorescent protein (eGFP) demonstrated high efficiency of transfection as well as transduction. Conclusion: Viral vectors constructed in this study could be used for investigation of osteogenesis.

Keywords

• Cloning
• MicoRNA
• MiR-148b
• Shuttle vectors
• Lentivirus