Overexpression of Ad5 precursor terminal protein accelerates recombinant adenovirus packaging and amplification in HEK-293 packaging cells.
Recombinant adenoviruses are one of the most common vehicles for efficient in vitro and in vivo gene deliveries. Here, we investigate whether exogenous precursor terminal protein (pTP) expression in 293 cells improves the efficiency of adenovirus packaging and amplification. We used a piggyBac transposon-based vector and engineered a stable 293 line that expresses high level of Ad5 pTP, designated as 293pTP. Using the AdBMP6-GLuc that expresses green fluorescent protein (GFP), BMP6 and Gaussia luciferase, we found that the infectivity of AdBMP6-GLuc viral samples packaged in 293pTP cells was titrated up to 19.3 times higher than that packaged in parental 293 cells. AdBMP6-GLuc viral samples packaged in 293pTP cells exhibited significantly higher transduction efficiency in 143B and immortalized mouse embryonic fibroblast (iMEF) cells, as assessed by fluorescence-activated cell sorting analysis of GFP-positive cells, the luciferase activity assay and BMP6-induced osteogenic marker alkaline phosphatase activities in iMEFs. When adenovirus amplification efficiency was analyzed, we found that 293pTP cells infected with AdBMP6-GLuc yielded up to 12.6 times higher titer than that in parental 293 cells, especially at lower multiplicities of infection. These results strongly suggest that exogenous pTP expression may accelerate the packaging and amplification of recombinant adenoviruses. Thus, the engineered 293pTP cells should be a superior packaging line for efficient adenovirus production.
1] Stem Cell Biology and Therapy Laboratory, Ministry of Education Key Laboratory for Pediatrics, and Chongqing Stem Cell Therapy and Engineering Center, The Children's Hospital of Chongqing Medical University, Chongqing, China [2] Department of Orthopaedic Surgery, Molecular Oncology Laboratory, University of Chicago Medical Center, Chicago, IL, USA [3] Ministry of Education Key Laboratory of Diagnostic Medicine and School of Clinical Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, China.
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Wu
1] Department of Orthopaedic Surgery, Molecular Oncology Laboratory, University of Chicago Medical Center, Chicago, IL, USA [2] Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences, and the Affiliated Hospital of Stomatology, Chongqing Medical University, Chongqing, China.
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Zhang
1] Department of Orthopaedic Surgery, Molecular Oncology Laboratory, University of Chicago Medical Center, Chicago, IL, USA [2] Departments of Cell Biology and Oncology of the Affiliated Southwest Hospital, the Third Military Medical University, Chongqing, China.
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Deng
1] Department of Orthopaedic Surgery, Molecular Oncology Laboratory, University of Chicago Medical Center, Chicago, IL, USA [2] Ministry of Education Key Laboratory of Diagnostic Medicine and School of Clinical Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, China.
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Li
1] Department of Orthopaedic Surgery, Molecular Oncology Laboratory, University of Chicago Medical Center, Chicago, IL, USA [2] Department of Laboratory Medicine, the Affiliated Hospital of Bingzhou Medical University, Yantai, China.
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Zhang
1] Department of Orthopaedic Surgery, Molecular Oncology Laboratory, University of Chicago Medical Center, Chicago, IL, USA [2] Ministry of Education Key Laboratory of Diagnostic Medicine and School of Clinical Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, China.
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Chen
1] Stem Cell Biology and Therapy Laboratory, Ministry of Education Key Laboratory for Pediatrics, and Chongqing Stem Cell Therapy and Engineering Center, The Children's Hospital of Chongqing Medical University, Chongqing, China [2] Department of Orthopaedic Surgery, Molecular Oncology Laboratory, University of Chicago Medical Center, Chicago, IL, USA.
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Wen
1] Department of Orthopaedic Surgery, Molecular Oncology Laboratory, University of Chicago Medical Center, Chicago, IL, USA [2] Departments of Cell Biology and Oncology of the Affiliated Southwest Hospital, the Third Military Medical University, Chongqing, China.
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Wang
1] Department of Orthopaedic Surgery, Molecular Oncology Laboratory, University of Chicago Medical Center, Chicago, IL, USA [2] Ministry of Education Key Laboratory of Diagnostic Medicine and School of Clinical Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, China.
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Zhang
1] Department of Orthopaedic Surgery, Molecular Oncology Laboratory, University of Chicago Medical Center, Chicago, IL, USA [2] Ministry of Education Key Laboratory of Diagnostic Medicine and School of Clinical Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, China.
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Yin
1] Department of Orthopaedic Surgery, Molecular Oncology Laboratory, University of Chicago Medical Center, Chicago, IL, USA [2] Department of Orthopaedic Surgery, the Affiliated Xiang-Ya Hospital of Central South University, Changsha, China.
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Liao
1] Department of Orthopaedic Surgery, Molecular Oncology Laboratory, University of Chicago Medical Center, Chicago, IL, USA [2] Department of Surgery, the Affiliated Zhongnan Hospital of Wuhan University, Wuhan, China.
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Zhang
1] Stem Cell Biology and Therapy Laboratory, Ministry of Education Key Laboratory for Pediatrics, and Chongqing Stem Cell Therapy and Engineering Center, The Children's Hospital of Chongqing Medical University, Chongqing, China [2] Department of Orthopaedic Surgery, Molecular Oncology Laboratory, University of Chicago Medical Center, Chicago, IL, USA.
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Zhang
1] Department of Orthopaedic Surgery, Molecular Oncology Laboratory, University of Chicago Medical Center, Chicago, IL, USA [2] Ministry of Education Key Laboratory of Diagnostic Medicine and School of Clinical Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, China.
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Yan
1] Stem Cell Biology and Therapy Laboratory, Ministry of Education Key Laboratory for Pediatrics, and Chongqing Stem Cell Therapy and Engineering Center, The Children's Hospital of Chongqing Medical University, Chongqing, China [2] Department of Orthopaedic Surgery, Molecular Oncology Laboratory, University of Chicago Medical Center, Chicago, IL, USA.
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Liu
Department of Orthopaedic Surgery, Molecular Oncology Laboratory, University of Chicago Medical Center, Chicago, IL, USA.
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Wu
1] Department of Orthopaedic Surgery, Molecular Oncology Laboratory, University of Chicago Medical Center, Chicago, IL, USA [2] School of Bioengineering, Chongqing University, Chongqing, China.
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Ye
1] Department of Orthopaedic Surgery, Molecular Oncology Laboratory, University of Chicago Medical Center, Chicago, IL, USA [2] Ministry of Education Key Laboratory of Diagnostic Medicine and School of Clinical Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, China.
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Deng
1] Stem Cell Biology and Therapy Laboratory, Ministry of Education Key Laboratory for Pediatrics, and Chongqing Stem Cell Therapy and Engineering Center, The Children's Hospital of Chongqing Medical University, Chongqing, China [2] Department of Orthopaedic Surgery, Molecular Oncology Laboratory, University of Chicago Medical Center, Chicago, IL, USA.
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Yang
Department of Orthopaedic Surgery, Molecular Oncology Laboratory, University of Chicago Medical Center, Chicago, IL, USA.
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Luu
Department of Orthopaedic Surgery, Molecular Oncology Laboratory, University of Chicago Medical Center, Chicago, IL, USA.
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Haydon
1] Stem Cell Biology and Therapy Laboratory, Ministry of Education Key Laboratory for Pediatrics, and Chongqing Stem Cell Therapy and Engineering Center, The Children's Hospital of Chongqing Medical University, Chongqing, China [2] Department of Orthopaedic Surgery, Molecular Oncology Laboratory, University of Chicago Medical Center, Chicago, IL, USA [3] Ministry of Education Key Laboratory of Diagnostic Medicine and School of Clinical Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, China.
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D000256:Adenoviridae / Q000235:genetics / Q000502:physiology*; D000257:Adenoviridae Infections / Q000821:virology; D000818:Animals; D045744:Cell Line, Tumor; D004622:Embryo, Mammalian / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000821:virology; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000821:virology; D051379:Mice; D010750:Phosphoproteins / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*; D011995:Recombination, Genetic; D014161:Transduction, Genetic; D014764:Viral Proteins / Q000378:metabolism*; D019065:Virus Assembly
D010750:Phosphoproteins; D011498:Protein Precursors; D014764:Viral Proteins; C085476:pTP protein, adenovirus