{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T16:14:10Z","timestamp":1785428050341,"version":"3.56.0"},"reference-count":24,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2026,6,22]],"date-time":"2026-06-22T00:00:00Z","timestamp":1782086400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100002855","name":"National Key R&D Program of China","doi-asserted-by":"publisher","award":["2023YFC2307900"],"award-info":[{"award-number":["2023YFC2307900"]}],"id":[{"id":"10.13039\/501100002855","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["82161138001"],"award-info":[{"award-number":["82161138001"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"award":["82161138001"],"award-info":[{"award-number":["82161138001"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["MPs"],"abstract":"<jats:p>Adenoviral vectors are widely used for gene therapy and vaccine development. To circumvent pre-existing immunity against commonly used human adenovirus type 5, vectors based on rare human serotype or animal adenoviruses have attracted increasing interest. Previously, we constructed vectors based on fowl adenovirus 4 (FAdV-4) and replaced the knob of FAdV-4 fiber2 with that of FAdV-1 fiber1 to generate FAdV4-CF1K vectors with enhanced transduction efficiency in human cells. In this study, we aimed to modify the packaging system to efficiently produce FAdV-4 vectors carrying transgenes toxic to viral replication. Chicken LMH cells failed to form colonies at low seeding densities. We collected used medium from LMH cell cultures and used it as a supplement to adapt LMH cells, generating the colony-competent subclone LMH-C3532. A lentiviral vector encoding a codon-optimized tetracycline repressor (tetR) was transduced into LMH-C3532 to establish a tetR-integrated cell line, LMH-tetR24. An adenoviral plasmid, pKFAV4-CF1K-CtG, was constructed in which a tetracycline operator (tetO)-bearing CMV promoter controlled GFP expression. The SwaI-flanked GFP in this plasmid was replaced with the HA gene from an H5N1 influenza virus to generate pKFAV4-CF1K-CtHA. Linearized adenoviral plasmids were transfected into LMH-tetR24 cells, and recombinant FAdV4-CF1K-CtG and FAdV4-CF1K-CtHA viruses were successfully rescued, amplified, and purified. When infected with FAdV4-CF1K-CtG at various multiplicities of infection (MOI), the progeny virus yield from LMH-tetR24 cells was 4\u201310 times higher than that from LMH-C3532 cells. For FAdV4-CF1K-CtHA, the yield difference between the two cell lines was even more pronounced, reaching 3\u20134 orders of magnitude. Overexpression of HA in LMH-C3532 cells negatively affected FAdV4-CF1K-CtHA replication, resulting in smaller and fewer plaques. In conclusion, by separately integrating tetR into packaging cells and TetO into the adenoviral plasmid, we established a system that can be routinely used to package FAdV-4 vectors. Notably, this system facilitates the propagation of FAdV-4 vectors carrying toxic transgenes.<\/jats:p>","DOI":"10.3390\/mps9030100","type":"journal-article","created":{"date-parts":[[2026,6,23]],"date-time":"2026-06-23T06:13:37Z","timestamp":1782195217000},"page":"100","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["An Efficient TetR\/TetO-Integrated Packaging System for Fowl Adenovirus 4 Vector Carrying Toxic Transgene"],"prefix":"10.3390","volume":"9","author":[{"given":"Qian-Wen","family":"Ma","sequence":"first","affiliation":[{"name":"NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhi","family":"Li","sequence":"additional","affiliation":[{"name":"NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China"},{"name":"School of Public Health, Baotou Medical College, Inner Mongolia University of Science and Technology, Baotou 014040, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhi-Chao","family":"Zhang","sequence":"additional","affiliation":[{"name":"NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1960-183X","authenticated-orcid":false,"given":"Xiao-Juan","family":"Guo","sequence":"additional","affiliation":[{"name":"NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6834-1341","authenticated-orcid":false,"given":"Xiao-Hui","family":"Zou","sequence":"additional","affiliation":[{"name":"NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tao","family":"Hung","sequence":"additional","affiliation":[{"name":"NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5767-401X","authenticated-orcid":false,"given":"Zhuo-Zhuang","family":"Lu","sequence":"additional","affiliation":[{"name":"NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,22]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"3395","DOI":"10.1002\/1873-3468.13700","article-title":"Lessons learned from adenovirus (1970\u20132019)","volume":"593","author":"Nemerow","year":"2019","journal-title":"FEBS Lett."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1016\/j.coviro.2021.03.006","article-title":"Adenovirus\u2014A blueprint for gene delivery","volume":"48","author":"Greber","year":"2021","journal-title":"Curr. Opin. Virol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"100432","DOI":"10.1016\/j.dmpk.2021.100432","article-title":"Adenovirus vector-based vaccine for infectious diseases","volume":"42","author":"Sakurai","year":"2022","journal-title":"Drug Metab. Pharmacokinet."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2514356","DOI":"10.1080\/21645515.2025.2514356","article-title":"Developing the next-generation of adenoviral vector vaccines","volume":"21","author":"Sampson","year":"2025","journal-title":"Hum. Vaccin. Immunother."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Trivedi, P.D., Byrne, B.J., and Corti, M. (2023). Evolving Horizons: Adenovirus Vectors\u2019 Timeless Influence on Cancer, Gene Therapy and Vaccines. Viruses, 15.","DOI":"10.3390\/v15122378"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"491","DOI":"10.1007\/s12275-024-00159-4","article-title":"Adenoviral Vector System: A Comprehensive Overview of Constructions, Therapeutic Applications and Host Responses","volume":"62","author":"Park","year":"2024","journal-title":"J. Microbiol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"3609","DOI":"10.1002\/1873-3468.13691","article-title":"State-of-the-art human adenovirus vectorology for therapeutic approaches","volume":"593","author":"Gao","year":"2019","journal-title":"FEBS Lett."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Alhashimi, M., Elkashif, A., Sayedahmed, E.E., and Mittal, S.K. (2021). Nonhuman Adenoviral Vector-Based Platforms and Their Utility in Designing Next Generation of Vaccines for Infectious Diseases. Viruses, 13.","DOI":"10.3390\/v13081493"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Yan, B., Zou, X., Liu, X., Zhao, J., Zhang, W., Guo, X., Wang, M., Lv, Y., and Lu, Z. (2020). User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome. Viruses, 12.","DOI":"10.3390\/v12030301"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Sun, Y., Zou, X., Guo, X., Yang, C., Hung, T., and Lu, Z. (2022). CELO Fiber1 Knob Is a Promising Candidate to Modify the Tropism of Adenoviral Vectors. Genes, 13.","DOI":"10.3390\/genes13122316"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"3109","DOI":"10.1046\/j.1432-1033.2003.03694.x","article-title":"Gene regulation by tetracyclines. Constraints of resistance regulation in bacteria shape TetR for application in eukaryotes","volume":"270","author":"Berens","year":"2003","journal-title":"Eur. J. Biochem."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1939","DOI":"10.1089\/hum.1998.9.13-1939","article-title":"Tetracycline repressor, tetR, rather than the tetR-mammalian cell transcription factor fusion derivatives, regulates inducible gene expression in mammalian cells","volume":"9","author":"Yao","year":"1998","journal-title":"Hum. Gene Ther."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"12126","DOI":"10.1038\/s41598-022-15526-7","article-title":"iCodon customizes gene expression based on the codon composition","volume":"12","author":"Diez","year":"2022","journal-title":"Sci. Rep."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1851","DOI":"10.1128\/JCM.43.4.1851-1857.2005","article-title":"Detection of human immunodeficiency virus type 1 DNA in dried blood spots by a duplex real-time PCR assay","volume":"43","author":"Luo","year":"2005","journal-title":"J. Clin. Microbiol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"495","DOI":"10.1038\/nprot.2009.22","article-title":"Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors","volume":"4","author":"Kutner","year":"2009","journal-title":"Nat. Protoc."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1023\/A:1009838416621","article-title":"Preparation of pure cell cultures by cloning","volume":"22","author":"McFarland","year":"2000","journal-title":"Methods Cell Sci."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"276","DOI":"10.1038\/s41587-020-0439-x","article-title":"The nf-core framework for community-curated bioinformatics pipelines","volume":"38","author":"Ewels","year":"2020","journal-title":"Nat. Biotechnol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"100130","DOI":"10.1016\/j.xpro.2020.100130","article-title":"Protocol for the Generation of Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells","volume":"1","author":"Nafria","year":"2020","journal-title":"STAR Protoc."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"100078","DOI":"10.1016\/j.xpro.2020.100078","article-title":"Protocol for the Maintenance of Quiescent Murine Hematopoietic Stem Cells","volume":"1","author":"Kobayashi","year":"2020","journal-title":"STAR Protoc."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1382","DOI":"10.1038\/sj.gt.3302780","article-title":"Optimization of the Tet-On system for regulated gene expression through viral evolution","volume":"13","author":"Zhou","year":"2006","journal-title":"Gene Ther."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Loew, R., Heinz, N., Hampf, M., Bujard, H., and Gossen, M. (2010). Improved Tet-responsive promoters with minimized background expression. BMC Biotechnol., 10.","DOI":"10.1186\/1472-6750-10-81"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"3311","DOI":"10.1038\/s41467-024-47592-y","article-title":"Engineering a synthetic gene circuit for high-performance inducible expression in mammalian systems","volume":"15","author":"Fusco","year":"2024","journal-title":"Nat. Commun."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"462","DOI":"10.1016\/j.redox.2017.03.002","article-title":"Fluorescent Proteins Such as eGFP Lead to Catalytic Oxidative Stress in Cells","volume":"12","author":"Ganini","year":"2017","journal-title":"Redox Biol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1002\/bit.27945","article-title":"Manufacturing a chimpanzee adenovirus-vectored SARS-CoV-2 vaccine to meet global needs","volume":"119","author":"Joe","year":"2022","journal-title":"Biotechnol. Bioeng."}],"container-title":["Methods and Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2409-9279\/9\/3\/100\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,23]],"date-time":"2026-06-23T07:48:03Z","timestamp":1782200883000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2409-9279\/9\/3\/100"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,22]]},"references-count":24,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2026,6]]}},"alternative-id":["mps9030100"],"URL":"https:\/\/doi.org\/10.3390\/mps9030100","relation":{},"ISSN":["2409-9279"],"issn-type":[{"value":"2409-9279","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,22]]}}}