Parameter Optimization In The Electroporation For Introduction Of Human Ribosomal Targeting Vector Into Hepatocyte

It is a great challenge to develop the gene therapy vector which can produce stable and efficient therapeutic effects and has no dangers of undesired immune response and insertion mutation. Viral vectors and nonviral vectors are two common methods. For the reason of safety, our lab has developed a novel nonviral vector-human ribosomal DNA-targeting vector: pHrneo, which is a human derived vector and has been showed a capacity of targeting hFVIII gene into HT1080 cell and site-specific integration. However, the low transfection efficiency into normal cells has still been a significant barrier for its application.Objectives: The human ribosomal DNA-targeting vector that contains green fluorescence protein (GFP) gene will be electroporated into human hepatocytes HL7702 to screen for a higher transfection efficiency by optimization of electroporation conditions and synchronization of cell cycle.Methods:1. The optimization of electroporation parametersSeveral key parameters, cell numbers and the volume of suspension, incubation conditions, the pulse voltage and capacitance and electroporation buffer (medium) were employed. According to these parameters, several groups of experiments were designed. The cell survival rate after electroporation was evaluated by trypan blue staining; GFP gene expression rate was detected by flow cytometry 48h after electroporation.2. The effect of cell synchronization on electroporationCell synchronization has been applied to improve the transfection efficiency and we synchronized cells in G2/M phase using hydroxyurea (HU), which is a cell synchronization drug. Then these cells and control group were applied to electroporation to analyze the effect of synchronizing cells in G2/M phase on electroporation.Results: Under the optimal conditions for transferring hrDNA targeting vector into HL7702 cell, 92.53±2.31% cell survival and 26.03±2.40% gene expression rates have been obtained. We have got more than 60% cells in the percentage of G2/M phase after cell synchronization and found that more than 90% cell survived and more than 60% cells expressed GFP gene after electroporation.Conclusions: As a conclusion, we obtained an optimized process that could stably result in more than 90% cell survival and 60% gene expression rates in electroporation, which would help better application of human ribosomal DNA-targeting vector to gene therapy.