Designing and Constructing the Lentiviral Vector Coding miRNA-499a and Transduction of Human Mesenchymal Stem Cells

Background and purpose: Lentiviral vectors (LVs) are suitable candidates for gene delivery to cells with stable and high-level of transgene expression in target cells. MicroRNAs (miRNAs, miRs) are non-protein coding, short (~22 nucleotides) and single-stranded RNAs that act as post-transcriptional regulators of gene expression, and are involved in various cellular processes, including proliferation, differentiation and apoptosis. Several studies have shown that miR-499a promotes cardiac differentiation in cardiac stem cells. So, the aim of our study was to construct lentiviruses carrying miRNA-499a. Specific sequences of miRNA-499a (3p and 5p) were designed and constructed. Then, miRNA-499a was cloned into lentiviral vector. Analytical digestion and nucleotide sequence analysis were performed to ensure successful introduction of the miRNA to the vector. Then, the lentiviral particles produced (miRNA-499a-3p and miRNA-499a-5p) were used for transduction of human bone marrow-derived mesenchymal stem cells (hBM-MSCs). Analytical digestion and sequence analysis confirmed the accuracy of the constructs. The high expression of eGFP represented the high efficiency of transfection and transduction. The lentiviral particles carrying miRNA-499a-3p/5p were made and could transduce hBM-MSCs. In this study we made lentiviral particles carrying miR-499a that could be used for differentiation of stem cells to cardiomyocytes.