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Objective To investigate the possibility of heterologous expression for apoAI, apoE and LCAT by skeletal muscle cells and secretion into blood and to develop a safe and convenient gene therapy method for atherosclerosis. Methods Viral and nonviral vectors containing apoAI, apoE or LCAT genes were constructed and transfected into myogenic cells in vitro or injected directed into mouse skeletal muscle. The expression efficiencies of these vectors were investigated by ELISA assay for human apoAI and apoE 3 and by the proteoliposome method for human LCAT. Genomic DNA was extracted from stable transduced myoblasts and analyzed for the presence of vector sequence by PCR amplifications. Immunocytochemistry assay was also performed to make an intuitionistic detection for the expression of transgene in myoblasts. Results All viral or nonviral vectors constructed in present study expressed the transgenes efficiently in mice skeletal muscles in vivo or cultured myoblasts in vitro. The transgene expression level of cells transfected with AAV based plasmid vectors were 2 4 times higher then that of cells transfected with conventional plasmid vectors. Additionally, cells transfected with AAV based bicistronic vector or tricistronic retroviral vector expressed both human apoAI and LCAT simultaneously. The sequences of retroviral or AAV based plasmid vectors were found to be retained in host cells after transfection when that of conventional plasmid vectors were lost. Furthermore, transduced myoblasts maintained the ability for heterologous expression of human apoAI and LCAT even after differentiation into myotubes. For cells transfected with retroviral vectors, stable transduced clones can be selected by G418 and continued to efficiently express human apoAI and LCAT for 3 months. Conclusion These finds indicated that mice skeletal muscles or cultured myoblasts transduced with viral or non viral vectors could efficiently express and secret human apoAI, apoE and LCAT. It suggested that the use of nonviral, adenoviral or AAV based vectors to directly inject into skeletal muscle or the use of polycistronic retroviral to genetically modify myoblasts ex vivo and then implantation back to skeletal muscle to high efficiently and long term express apoAI, apoE and LCAT in vivo might be a safe and feasible strategy to prevent or reduce the formation of atherosclerotic lesions.
Objective To investigate the possibility of heterologous expression for apoAI, apoE and LCAT by skeletal muscle cells and secretion into blood and to develop a safe and convenient gene therapy method for atherosclerosis. Methods Viral and nonviral vectors containing apoAI, apoE or LCAT genes were constructed and The expression efficiencies of these vectors were investigated by ELISA assay for human apoAI and apoE 3 and by the proteoliposome method for human LCAT. Genomic DNA was extracted from stable transduced myoblasts and analyzed for the presence of vector sequence by PCR amplifications. Immunocytochemistry assay was also performed to make an intuitionistic detection for the expression of transgene in myoblasts. Results All viral or nonviral vectors constructed in present study showed the transgenes efficiently in mice skeletal muscles in vivo or cultured myoblasts in vitro. The transge ne expression level of cells transfected with AAV based plasmid vectors were 2 4 times higher then that of cells transfected with conventional plasmid vectors. Additionally, cells transfected with AAV based bicistronic vector or tricistronic retroviral vector expressed both human apoAI and LCAT simultaneously. The sequences of retroviral or AAV based plasmid vectors were found to be retained in host cells after transfection when that of conventional plasmid vectors were lost. Furthermore, transduced myoblasts maintained the ability for heterologous expression of human apoAI and LCAT even after differentiation into myotubes. For cells transfected with retroviral vectors, stable transduced clones can be selected by G418 and continued to efficiently express human apoAI and LCAT for 3 months. Conclusion These finds indicates that mice skeletal muscles or cultured myoblasts transduced with viral or non viral vectors could effectively express and secret human apoAI, apoE and LCAT. It suggested that the use of nonviral, adenoviral or AAV based vectors to directly injected into skeletal muscle or the use of polycistronic retroviral to genetically modify myoblasts ex vivo and then implantation back to skeletal muscle to high efficiently and long term express apoAI, apoE and LCAT in vivo might be a safe and feasible strategy to prevent or reduce the formation of atherosclerotic lesions.