Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology · 2017
Engineering the Rapid Adenovirus Production and Amplification (RAPA) Cell Line to Expedite the Generation of Recombinant Adenoviruses.
СтатьяНазвание и аннотация: Перевод в очереди
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Wei Qiang, Fan Jiaming, Liao Junyi, Zou Yulong, Song Dongzhe, Liu Jianxiang, Cui Jing, Liu Feng, Ma Chao, Hu Xue, Li Li, Yu Yichun, Qu Xiangyang, Chen Liqun, Yu Xinyi, Zhang Zhicai, Zhao Chen, Zeng Zongyue, Zhang Ruyi, Yan Shujuan, Wu Xingye, Shu Yi, Reid Russell R, Lee Michael J, Wolf Jennifer Moritis, He Tong-Chuan
Аннотация
BACKGROUND/AIMS: While recombinant adenoviruses are among the most widely-used gene delivery vectors and usually propagated in HEK-293 cells, generating recombinant adenoviruses remains time-consuming and labor-intense. We sought to develop a rapid adenovirus production and amplification (RAPA) line by assessing human Ad5 genes (E1A, E1B19K/55K, pTP, DBP, and DNA Pol) and OCT1 for their contributions to adenovirus production.
METHODS: Stable transgene expression in 293T cells was accomplished by using piggyBac system. Transgene expression was determined by qPCR. Adenoviral production was assessed with titering, fluorescent markers and/or luciferase activity. Osteogenic activity was assessed by measuring alkaline phosphatase activity.
RESULTS: Overexpression of both E1A and pTP led to a significant increase in adenovirus amplification, whereas other transgene combinations did not significantly affect adenovirus amplification. When E1A and pTP were stably expressed in 293T cells, the resultant RAPA line showed high efficiency in adenovirus amplification and production. The produced AdBMP9 infected mesenchymal stem cells with highest efficiency and induced most effective osteogenic differentiation. Furthermore, adenovirus production efficiency in RAPA cells was dependent on the amount of transfected DNA. Under optimal transfection conditions high-titer adenoviruses were obtained within 5 days of transfection.
CONCLUSION: The RAPA cells are highly efficient for adenovirus production and amplification.
METHODS: Stable transgene expression in 293T cells was accomplished by using piggyBac system. Transgene expression was determined by qPCR. Adenoviral production was assessed with titering, fluorescent markers and/or luciferase activity. Osteogenic activity was assessed by measuring alkaline phosphatase activity.
RESULTS: Overexpression of both E1A and pTP led to a significant increase in adenovirus amplification, whereas other transgene combinations did not significantly affect adenovirus amplification. When E1A and pTP were stably expressed in 293T cells, the resultant RAPA line showed high efficiency in adenovirus amplification and production. The produced AdBMP9 infected mesenchymal stem cells with highest efficiency and induced most effective osteogenic differentiation. Furthermore, adenovirus production efficiency in RAPA cells was dependent on the amount of transfected DNA. Under optimal transfection conditions high-titer adenoviruses were obtained within 5 days of transfection.
CONCLUSION: The RAPA cells are highly efficient for adenovirus production and amplification.