| Summary: | 碩士 === 長庚大學 === 醫學生物技術研究所 === 96 === Lentiviruses are the type of retrovirus that can infect both dividing and nondividing cells. Lentiviruses can be used to provide highly effective gene transfer as well as gene therapy. Despite of success, traditional gene cloning for viral vector included choosing suitable restriction enzyme and confirming the gene orientation remains a major obstacle. Recently, scientists developed a new gene cloning technique called Gateway Recombination System. This system allowed transfer of DNA fragments by specific recombination site between different cloning vectors without restriction endonucleases and ligase while maintaining the reading frame. Therefore, we employed Gateway Recombination System to establish a convenient lentiviral vector technical platform. For the application of gene therapy, choice of promoter affects the gene expression of the vector. There are many kinds of RNA polymerase II promoter including CMV, UBC, and inducible CMV-TO promoter. These promoters have their own characteristic. CMV promoter is a viral promoter and is often thought of as a constitutively active unregulated promoter. Human ubiquitin C (UBC) promoter is a endogenous promoter and highly conserved in all eukaryotic cell. So it can constitutively express gene in mammalian cell. And CMV-TO promoter is a promoter based on tetracycline repressor (TetR) inducible system. To accomplish our goal, we studied: (1) to characterize gene expression efficiency in the lentiviral vector with gateway recombination, (2) to investigate gene expression efficiency in the lentiviral vector with different promoter. Our results shown: We have already found the best reaction condition of gateway recombination and successfully exchanged gene sequence into improved viral vector. In microRNA-based RNA interference miR-shRNA system, we found that compared with viral vector containing miR30 shRNA targeted luciferase gene in miR30 cassette, the viral vector with miR155 shRNA in miR155 cassette has better inhibition efficiency of luciferase activity. Furthermore, we have successfully adopted the Gateway Recombination System for efficient route for subclone different promoters into lentiviral systems. Moreover, we also found that the lentiviral vectors containing both human ubiquitin C promoter and WPRE fragment has higher gene expression efficiency when compared with other promoters. In conclusion, we have successfully established an extremely fast and convenient viral vector cloning technique and higher gene expression efficiency in lentiviral systems. This system would provide us an opportunity to improve usefulness of lentiviral vector for gene therapy in near future.
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