Qiang Wei1,2, Jiaming Fan1,2, Junyi Liao1,2, Yulong Zou1,2, Dongzhe Song2,3, Jianxiang Liu2,4, Jing Cui1,2, Feng Liu1,2, Chao Ma2,5, Xue Hu1,2, Li Li2,6, Yichun Yu2,7, Xiangyang Qu1,2, Liqun Chen1,2, Xinyi Yu1,2, Zhicai Zhang2,4, Chen Zhao1,2, Zongyue Zeng1,2, Ruyi Zhang1,2, Shujuan Yan1,2, Xingye Wu1,2, Yi Shu1,2, Russell R Reid8, Michael J Lee2, Jennifer Moritis Wolf2, Tong-Chuan He1,2. 1. Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, China. 2. Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois, USA. 3. Department of Conservative Dentistry and Endodontics, West China School of Stomatology, Sichuan University, Chengdu, China. 4. Department of Orthopaedic Surgery, Union Hospital of Tongji Medical College, Huazhong University of Science & Technology, Wuhan, China. 5. Department of Neurosurgery, the Affiliated Zhongnan Hospital of Wuhan University, Wuhan, China. 6. Department of Biomedical Engineering, School of Bioengineering, Chongqing University, Chongqing, China. 7. Department of Emergency Medicine, Beijing Hospital, Beijing, China. 8. Department of Surgery, Section of Plastic Surgery, The University of Chicago Medical Center, Chicago, Illinois, USA.
Abstract
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.
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.
Authors: Xia Zhao; Bo Huang; Hao Wang; Na Ni; Fang He; Qing Liu; Deyao Shi; Connie Chen; Piao Zhao; Xi Wang; William Wagstaff; Mikhail Pakvasa; Andrew Blake Tucker; Michael J Lee; Jennifer Moriatis Wolf; Russell R Reid; Kelly Hynes; Jason Strelzow; Sherwin H Ho; Tengbo Yu; Jian Yang; Le Shen; Tong-Chuan He; Yongtao Zhang Journal: Am J Transl Res Date: 2021-05-15 Impact factor: 4.060