Background:This study was designed to prove the superiority of ACP vector containing ITR, and to establish animal model quantifying angiogenesis in vivo.
Methods:hVEGF121, therapeutic gene, was inserted to various vectors (pcDNA3.1, pcDNA 3.2, pActin, pDesm, pACP vector), and these vectors were transfected to various cells using FuGENE6. We cultured for 48hrs, and then quantified amounts of hVEGF121 of supernatants by ELISA. The long-term transfection was assessed for 14 days. Optimal condition of transfection was evaluated by change of the ratio of DNA to FuGENE6, amount of DNA, and confluence of cells. ACP-hVEGF121 was transfected to C2C12 and these transfected C2C12 cells were mixed with Matrigel, and then injected to C3H mouse subcutaneously. Seven days later, hemoglobin assay and pathology of Matrigel were reviewed for angiogenesis.
Results:The level of hVEGF121 gene expression using pACP vector was significantly higher than those of others. In 2 weeks culture study, pACP vector showed the highest gene expression and produced VEGF until 2 weeks. The highest gene expression was obtained when the concentration of DNA was 7 microgram, the confluence was up to 80% and the ratio of DNA to FuGENE6 was 1:3. The hemoglobin level in Matrigel of VEGF group was significantly higher than the one of the control group, and active angiogenesis was noted in the VEGF group.
Conclusions:pACP vector might be an efficient vector for angiogenic gene delivery, and animal model using Matrigel and transfected C2C12 cell could be a useful tool for quantitative angiogenesis assay.
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