Abstract
We have compared the efficacy and toxicity of the cytomegalovirus (CMV) promoter and the Rous sarcoma virus (RSV) promoter for expressing genes for adenovirus-mediated gene transfer to mesenchymal stem cells (MSCs). Mouse MSCs (mMSCs) were isolated from the femurs and tibias of 6-week old, female BALB/c mice and ex vivo expanded in MEM-alpha containing 20% fetal bovine serum, 100 u/ml penicillin, 100 μg/ml streptomycin, 250 ng/ml amphotericin B, and 2 mM L-glutamine. To transduce mMSCs with adenovirus, the cells were plated at a density of 10,000 cells/cm2 in 6-well plates and cultured overnight. The cells were counted and then exposed to fresh culture medium containing adenovirus at 0 to 2,000 multiplicities of infection (MOI) for 48 h. Two adenoviral vectors with CMV promoter (Adv/CMV) and two adenoviral vectors with RSV promoter (Adv/RSV) were used in this study. Transduction efficiency and cell survival were then determined. The Adv/CMVs, i.e. AdvCMVlacZ and AdvCMVIL-10, were more potent in terms of transduction efficiency and transgene expression than their corresponding Adv/RSVs, i.e. AdvRSVlacZ and AdvRSVIL-10. However, both vectors exhibit a dose-dependent relationship between the adenoviral vector MOI and the percentage of transgene-expressing cells. Furthermore, no toxicity was observed in Adv/CMV or Adv/RSV transduced mMSCs.
183. Development of Ex-Vivo Gene Therapy Procedures for Central Nervous System Diseases, Based on Homologous Recombination in Human Neural Stem Cells