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Transduction of various mammalian bone marrow--derived mesenchymal stem cells by baculovirus

Liu Zhengshan, Zhang Cheng, Lu Xilin, Li Yong, Xu Yongfeng, Xiong Fu, Feng Shanwei, Li Ling

Department of Neurology,the First Affiliated Hospital,Sun Yat-Sen University.Guangzhou 510080,Guangdong;China;Department of Medical Genetics,Southern Medical University.Guangzhou 510515,Guangdong

Abstract

The use of stem cells will lead to novel treatments for a wide range of diseases due to the properties of self-renewing, pluripotent, and undifferentiated state, and the stem cells are usually genetically modified for cell and gene therapy. If the baculovirus, as a new gene vector, can be effectively transduced into various mammalian bone marrow-derived mesenchymal stem cells (BMSCs) in vitro, it will be a better gene vector to genetically modify the stem cells. The aim of the present study was to investigate the transduction efficiency of baculovirus into various mammalian BMSCs, which expressed a reporter gene encoding enhanced green fluorescent protein (EGFP) under a cytomegalovirus immediate early (CMV-IE) promoter. The BMSCs in mouse, rat, porcine, rhesus, and human were cultured primarily in vitro. After more than three passages, the mammalian BMSCs were seeded into dishes and cultured in a humidified incubator at 37 ℃ with 5% CO_(2). When the cells reached about 80% confluence, the complete medium was removed by aspiration. The cells were transduced with baculovirus at a multiplicity of infection (MOI) of 200 vector genomes/cell with 500 #mu#L of PBS at 25 ℃ for 4 h. At the end of baculovirus transduction, cells were washed and incubated with 2 mL complete medium, and baculovirus-transduced mammalian BMSCs were cultured in a humidified incubator for 2 d. Then, the invert fluorescent microscope was used to observe GFP expression in different mammalian BMSCs, flow cytometry was used to detect the transduction efficiency of baculovirus in various mammalian BMSCs. After more than three passages, the BMSCs in mouse, rat, porcine, rhesus, and human showed a homogeneous spindle-shaped morphology. Compared with the BMSCs in mouse, rat and porcine, the invert fluorescent microscope observations showed that there were more BMSCs in rhesus and human expressing GFP, which were transduced with baculovirus, and greater mean fluorescence intensity in rhesus and human BMSCs was observed. The baculovirus could efficiently transduce the BMSCs in mouse, rat, porcine, rhesus, and human, and the transduction efficiency of baculovirus into BMSCs in mouse, rat, porcine, rhesus, and human was (20.21±3.02)%, (22.51±4.48)%, (39.13±5.79)%, (71.16±5.36)% and (70.67±3.74)%, respectively. In conclusion, baculovirus displays different transduction efficiency into various mammalian BMSCs. Due to the high transduction efficiency for primate and human BMSCs, baculovirus is possibly a more suitable gene vector to genetically modify BMSCs in human and primates.

Key words: bone marrow-derived mesenchymal stem cells;baculovirus;transduction;green fluorescent protein

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Citing This Article:

Liu Zhengshan, Zhang Cheng, Lu Xilin, Li Yong, Xu Yongfeng, Xiong Fu, Feng Shanwei, Li Ling. Transduction of various mammalian bone marrow--derived mesenchymal stem cells by baculovirus. Acta Physiol Sin 2008; 60 (3): 431-436 (in Chinese with English abstract).