Engineering the Rapid Adenovirus Production and Amplification (RAPA) Cell Line to Expedite the Generation of Recombinant Adenoviruses

Wei, Qiang; Fan, Jiaming; Liao, Junyi; Zou, Yulong; Song, Dongzhe; Liu, Jianxiang; Cui, Jing; Liu, Feng; C, Ma; Hu, Xin; 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

doi:10.1159/000475909

Cited by 50 publications

(52 citation statements)

References 41 publications

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“…293pTP and RAPA cells were previously characterized. 22 , 23 All of these cell lines were maintained in the completed Dulbecco's modified Eagle medium (DMEM) as described. 13 , 24 , 25 , 26 The parental SV40 T-antigen temperature sensitive-mutant immortalized mouse podocytes (designated as tsPC cells) were kindly provided by Dr. Yan-Chun Li of The University of Chicago and maintained under permissive conditions at 33 °C with RPMI 1640 containing 10% FBS, 100 U/ml γ-IFN, and 100 U/ml penicillin/streptomycin as described.…”

Section: Methodsmentioning

confidence: 99%

“…28 , 29 , 30 , 31 , 32 Briefly, the coding regions of mouse TGFβ1 and FLP recombinase were PCR amplified and cloned into an adenoviral shuttle vector and subsequently used to generate recombinant adenoviruses in HEK-293, 293pTP or RAPA cells. 22 , 23 The resulting adenoviruses were designated as Ad-TGFβ1 and Ad-FLP, both of which also express GFP as the marker for monitoring infection efficiency. Analogous adenovirus expressing only GFP or RFP (Ad-GFP or Ad-RFP) was used as a mock control as described.…”

Section: Methodsmentioning

confidence: 99%

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Establishment and functional characterization of the reversibly immortalized mouse glomerular podocytes (imPODs)

Chen

et al. 2018

Genes & Diseases

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Glomerular podocytes are highly specialized epithelial cells and play an essential role in establishing the selective permeability of the glomerular filtration barrier of kidney. Maintaining the viability and structural integrity of podocytes is critical to the clinical management of glomerular diseases, which requires a thorough understanding of podocyte cell biology. As mature podocytes lose proliferative capacity, a conditionally SV40 mutant tsA58-immortalized mouse podocyte line (designated as tsPC) was established from the Immortomouse over 20 years ago. However, the utility of the tsPC cells is hampered by the practical inconvenience of culturing these cells. In this study, we establish a user-friendly and reversibly-immortalized mouse podocyte line (designated as imPOD), on the basis of the tsPC cells by stably expressing the wildtype SV40 T-antigen, which is flanked with FRT sites. We show the imPOD cells exhibit long-term high proliferative activity, which can be effectively reversed by FLP recombinase. The imPOD cells express most podocyte-related markers, including WT-1, Nephrin, Tubulin and Vinculin, but not differentiation marker Synaptopodin. The imPOD cells do not form tumor-like masses in vivo. We further demonstrate that TGFβ1 induces a podocyte injury-like response in the FLP-reverted imPOD cells by suppressing the expression of slit diaphragm-associated proteins P-Cadherin and ZO-1 and upregulating the expression of mesenchymal markers, α-SMA, Vimentin and Nestin, as well as fibrogenic factors CTGF and Col1a1. Collectively, our results strongly demonstrate that the newly engineered imPOD cells should be a valuable tool to study podocyte biology both under normal and under pathological conditions.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Establishment and functional characterization of the reversibly immortalized mouse glomerular podocytes (imPODs)

Chen

et al. 2018

Genes & Diseases

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“…22,23 Primary mouse hepatocytes were isolated from 4-week-old C57BL/6J mice using the type I collagenase/liver perfusion protocol as described. 22,23 Primary mouse hepatocytes were isolated from 4-week-old C57BL/6J mice using the type I collagenase/liver perfusion protocol as described.…”

Section: Cell Culture and Chemicalsmentioning

confidence: 99%

“…HEK-293 derivatives 293pTP and RAPA cells were previously described. 22,23 Primary mouse hepatocytes were isolated from 4-week-old C57BL/6J mice using the type I collagenase/liver perfusion protocol as described. 24,25 Mouse hepatocyte line iHPx cells are reversibly immortalized mouse E12.5 hepatocytes as described previously.…”

Section: Cell Culture and Chemicalsmentioning

confidence: 99%

Long non‐coding RNA (lncRNA) H19 induces hepatic steatosis through activating MLXIPL and mTORC1 networks in hepatocytes

Wang

Cao

Shu

et al. 2019

J Cellular Molecular Medi

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Liver plays an essential role in regulating lipid metabolism, and chronically disturbed hepatic metabolism may cause obesity and metabolic syndrome, which may lead to non-alcoholic fatty liver disease (NAFLD). Increasing evidence indicates long noncoding RNAs (lncRNAs) play an important role in energy metabolism. Here, we investigated the role of lncRNA H19 in hepatic lipid metabolism and its potential association with NAFLD. We found that H19 was up-regulated in oleic acid-induced steatosis and during the development of high-fat diet (HFD)-induced NAFLD. Exogenous overexpression of H19 in hepatocytes induced lipid accumulation and up-regulated the expression of numerous genes involved in lipid synthesis, storage and breakdown, while silencing endogenous H19 led to a decreased lipid accumulation in hepatocytes. Mechanistically, H19 was shown to promote hepatic steatosis by up-regulating lipogenic transcription factor MLXIPL. Silencing Mlxipl diminished H19-induced lipid accumulation in hepatocytes. Furthermore, H19-induced lipid accumulation was effectively inhibited by PI3K/mTOR inhibitor PF-04691502. Accordingly, H19 overexpression in hepatocytes up-regulated most components of the mTORC1 signalling axis, which were inhibited by silencing endogenous H19. In vivo hepatocyte implantation studies further confirm that H19 promoted hepatic steatosis by up-regulating both mTORC1 signalling axis and MLXIPL transcriptional network. Collectively, these

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“… 135 More recently, we have engineered the RAPA cell line for rapid adenovirus production and amplification. 136 By assessing human Ad5 genes (E1A, E1B19K/55K, pTP, DBP, and DNA Pol) and host factor OCT1 for their contributions to adenovirus production, we demonstrated that 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.…”

Section: Approaches To the Efficient Productions Of Recombinant Adenomentioning

confidence: 86%

Adenovirus-mediated gene delivery: Potential applications for gene and cell-based therapies in the new era of personalized medicine

et al. 2017

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With rapid advances in understanding molecular pathogenesis of human diseases in the era of genome sciences and systems biology, it is anticipated that increasing numbers of therapeutic genes or targets will become available for targeted therapies. Despite numerous setbacks, efficacious gene and/or cell-based therapies still hold the great promise to revolutionize the clinical management of human diseases. It is wildly recognized that poor gene delivery is the limiting factor for most in vivo gene therapies. There has been a long-lasting interest in using viral vectors, especially adenoviral vectors, to deliver therapeutic genes for the past two decades. Among all currently available viral vectors, adenovirus is the most efficient gene delivery system in a broad range of cell and tissue types. The applications of adenoviral vectors in gene delivery have greatly increased in number and efficiency since their initial development. In fact, among over 2000 gene therapy clinical trials approved worldwide since 1989, a significant portion of the trials have utilized adenoviral vectors. This review aims to provide a comprehensive overview on the characteristics of adenoviral vectors, including adenoviral biology, approaches to engineering adenoviral vectors, and their applications in clinical and preclinical studies with an emphasis in the areas of cancer treatment, vaccination and regenerative medicine. Current challenges and future directions regarding the use of adenoviral vectors are also discussed. It is expected that the continued improvements in adenoviral vectors should provide great opportunities for cell and gene therapies to live up to its enormous potential in personalized medicine.

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Engineering the Rapid Adenovirus Production and Amplification (RAPA) Cell Line to Expedite the Generation of Recombinant Adenoviruses

Cited by 50 publications

References 41 publications

Establishment and functional characterization of the reversibly immortalized mouse glomerular podocytes (imPODs)

Establishment and functional characterization of the reversibly immortalized mouse glomerular podocytes (imPODs)

Long non‐coding RNA (lncRNA) H19 induces hepatic steatosis through activating MLXIPL and mTORC1 networks in hepatocytes

Adenovirus-mediated gene delivery: Potential applications for gene and cell-based therapies in the new era of personalized medicine

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