Preliminary Exploration Of Targeting To The MSCs With The RDNA Targeting Vector Carrying Tumor Suppressor Gene IL-24

With the development of molecular biology, human’s understanding of recombinant DNA technology become mature gradually, As a new methods for cancer treatment,tumor gene therapy arises at the historic moment. In tumor gene therapy, the choice of vector and transport cells is particularly critical.Our lab has been committed to the research of safe and efficient non viral vector.In the prophase study we has successfully built a vector for cancer gene therapy:pHrneo-IL24. On the choice of target cells, MSCs are capable of migrating to tumor location and easy to separate, and could differentiate into multiple cell.Some reports show that MSC has been applied in tumor pre-clinical testing. Targeted gene modification of MSCs can more promote its curative effect in tumor gene therapy, but the genetic modification of MSC has always been a technical problem. The presence of the nucleic acid engineering enzyme TALEN provides a tool to break through this difficult problem. Therefore, this research intends to combine TALENickases (our lab established TALEN efficient mutants) with IL-24ribosomal gene region of targeted vector pHrneo-IL24for gene targeting MSC cells. Excepted to get fixed-point integration of cloning which can express biological activity of IL-24protein.Objective:We use the pHrneo-IL24vector with TALENickases targeting MSCs.We expect to get cell lines can expression of IL-24stablely.Methods:1. Isolation and culture of MSCs:separation of bone marrow mononuclear cells from bone marrow specimens, and separate the mesenchymal stem cells (MSCs) by adherent culture.2. Flow cytometry text MSCs surface markers.3. TALENickases and pHrneo-IL24plasmid co-transfer to MSCs, observing the GFP fusion protein expression by microscope and analysis of transfection efficiency.4, Cells culture in two lines, one line joining G418to screening point integration of cloning, another line amplification culture directly.5. Extract targeting cell gDNA, to text the fixed-point integration.6. Collecting cell supernatant, ELISA to detect IL-24protein expression of cells.Results:1. MSCs separate from bone marrow specimens are short spindle and appearance neat. MSCs is fibrous after several times of passage.Results show that the match various surface markers of MSCs;2. MSCs microscopically have green fluorescent expression after nuclear transfect. A large number of cell death after G418screening. There is no integration of clonal cells.3. The amplification of the after transfection of MSCs without G418screening, extract its gDNA, PCR detected exists a certain amount of fixed-point integration cells.4. Collect mixed cell supernatant,17.459ng/ml IL-24protein expression can be detected by ELISA.Conclusion:Successfully separation of MSCs. Preliminary validation TALENickases with pHrneo IL-24vector can gene targeting to MSCs and express protein of IL-24.