Efficiency of different recombinant viral vectors in the transduction of bone marrow mesenchymal stem cells a nd exogenous gene expression / 中国组织工程研究

ABSTRACT

BACKGROUND: Genetically engineered cells have been used in the replacement therapy and gene therapy. However, how to select proper donor cells, target cells, and corresponding viral vectors is the most difficult in the therapy.OBJECTIVE: To compare the transduction efficiency of recombinant adenovirus Ad5 and AdSF35, adeno-associated virus rAAV1/2 and rAAV2, and lentivirus LV in bone marrow mesenchymal stem cells (BMSCs) and exogenous gene expression level so as to select the vectors, which can efficiently transduce BMSCs.DESIGN, TIME AND SETTING: Gene engineering controlled observation, performed in the Central Laboratory, Shanghai First People's Hospital between October 2006 and March 2007.MATERIALS Ten Sprague Dawley rats of clean grade were used to prepare BMSCs. All recombinant viral vectors carded enhanced green fluorescent protein (EGFP) report gene. Ad5 was prepared by the Central Laboratory, Shanghai First People's Hospital. Ad5F35 was gifted by professor Qian Qi-jun from the Second Military Medical University of Chinese PLA. rAAV2 and rAAVI/2 were the products of Benyuan Zhengyang Gene Technique Co.,Ltd. LV was gifted by professor Cuo Li-be from Shanghai Institute of Biochemistry and Cytobiology, Chinese Academy of Sciences.METHODS: Rat BMSCs were in vitro isolated and cultured by density gradient centrifugation. BMSCs of passage 4 were inoculated into 24-well plate at lxlO5/well. One day later, ceils adhered to the wall and allocated to 5 groups. Ad5-EGFP [10, 100,1 000 multiplicity of infection (MOI)], Ad5F35-EGFP (10,10, 1 000 MOI), rAAVI/2-EGFP (1×104,1x10× vg), rAAV2-EGFP(1×104, 1x105vg), and LV-EGFP (30 TU) were respectively added into the 5 groups. BMSCs were transduced for 2 days with Ad virus and separately for 6 days with rAAV and LV virus.MAIN OUTCOME MEASURES: EGFP-positive expression and fluorescence intensity.RESULTS: After twenty-four hours of Ad5-EGFP transduction, EGFP-positive cells were visible under the microscope. With virus dose increasing, EGFP-positive cells increased and fluorescence intensity strengthened. Twelve days later, EGFP-positive cells gradually reduced and fluorescence intensity weakened. For Ad5F35-EGFP, its transduction was basically similar to Ad5-EGFP, but EGFP-positive cell number and fluorescence intensity were increased. After 6 days of rAAV1/2-EGFP or rAAV2-EGFP transduction, EGFP-posidve cell number and fluorescence intensity were decreased. For LV-EGFP transduction, a small amount of EGFP-positive cells could be visible on the second day, and then EGFP-positive ceils and fluorescence intensity were gradually increased until the sixth day.CONCLUSION: Ad5, Ad5F35 and LV could effectively transduce BMSCs cultured in vitro and express exogenous gene.Furthermore, transduction efficiency was correlated with virus dose in dose-dependent manner, rAAVI/2 and rAAV2 had poor/in vitro transduction efficiency.