{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,23]],"date-time":"2026-01-23T11:22:32Z","timestamp":1769167352320,"version":"3.49.0"},"reference-count":38,"publisher":"Wiley","issue":"10","license":[{"start":{"date-parts":[[2022,10,27]],"date-time":"2022-10-27T00:00:00Z","timestamp":1666828800000},"content-version":"vor","delay-in-days":26,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2022,10]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>The Sleeping Beauty (SB) transposon system is an efficient non\u2010viral tool for gene transfer into a variety of cells, including human cells. Through a cut\u2010and\u2010paste mechanism, your favorite gene (YFG) is integrated into AT\u2010rich regions within the genome, providing stable long\u2010term expression of the transfected gene. The SB system is evolving and has become a powerful tool for gene therapy. There are no safety concerns using this system, the handling is easy, and the time required to obtain a stable cell line is significantly reduced compared to other systems currently available. Here, we present a novel application of this system to generate, within 8 days, a stable producer HEK293T cell line capable of constitutively delivering enveloped virus\u2010like particles (eVLPs) for vaccination. We provide step\u2010by\u2010step protocols for generation of the SB transposon constructs, transfection procedures, and validation of the produced eVLPs. We next describe a method to pseudotype the constitutively produced eVLPs using the Spike protein derived from the SARS\u2010CoV\u20102 virus (by coating the eVLP capsid with the heterologous antigen). We also describe optimization methods to scale up the production of pseudotyped eVLPs in a laboratory setting (from 100 \u00b5g to 5 mg). \u00a9 Published 2022. This article is a U.S. Government work and is in the public domain in the USA.<\/jats:p><jats:p><jats:bold>Basic Protocol 1<\/jats:bold>: Generation of the SB plasmids<\/jats:p><jats:p><jats:bold>Basic Protocol 2<\/jats:bold>: Generation of a stable HEK293T cell line constitutively secreting MLV\u2010based eVLPs<\/jats:p><jats:p><jats:bold>Basic Protocol 3<\/jats:bold>: Evaluation of the SB constructs by immunofluorescence assay<\/jats:p><jats:p><jats:bold>Basic Protocol 4<\/jats:bold>: Validation of eVLPs by denaturing PAGE and western blot<\/jats:p><jats:p><jats:bold>Alternate Protocol 1<\/jats:bold>: Analysis of SARS\u2010CoV\u20102 Spike protein oligomerization using blue native gel electrophoresis and western blot<\/jats:p><jats:p><jats:bold>Alternate Protocol 2<\/jats:bold>: Evaluation of eVLP quality by electron microscopy (negative staining)<\/jats:p><jats:p><jats:bold>Basic Protocol 5<\/jats:bold>: Small\u2010scale production of eVLPs<\/jats:p><jats:p><jats:bold>Alternate Protocol 3<\/jats:bold>: Large\u2010scale production of eVLPs (up to about 1 to 3 mg VLPs)<\/jats:p><jats:p><jats:bold>Alternate Protocol 4<\/jats:bold>: Large\u2010scale production of eVLPs (up to about 3 to 5 mg VLPs)<\/jats:p><jats:p><jats:bold>Support Protocol<\/jats:bold>: Quantification of total protein concentration by Bradford assay<\/jats:p>","DOI":"10.1002\/cpz1.575","type":"journal-article","created":{"date-parts":[[2022,10,27]],"date-time":"2022-10-27T12:35:33Z","timestamp":1666874133000},"source":"Crossref","is-referenced-by-count":2,"title":["Using the Sleeping Beauty (SB) Transposon to Generate Stable Cells Producing Enveloped Virus\u2010Like Particles (eVLPs) Pseudotyped with SARS\u2010CoV\u20102 Proteins for Vaccination"],"prefix":"10.1002","volume":"2","author":[{"given":"Viviana","family":"Pszenny","sequence":"first","affiliation":[{"name":"Molecular Parasitology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases National Institutes of Health  Bethesda Maryland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Erick","family":"Tjhin","sequence":"additional","affiliation":[{"name":"Molecular Parasitology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases National Institutes of Health  Bethesda Maryland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Eliza V.C.","family":"Alves\u2010Ferreira","sequence":"additional","affiliation":[{"name":"Molecular Parasitology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases National Institutes of Health  Bethesda Maryland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Stephanie","family":"Spada","sequence":"additional","affiliation":[{"name":"Molecular Parasitology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases National Institutes of Health  Bethesda Maryland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fadila","family":"Bouamr","sequence":"additional","affiliation":[{"name":"Molecular Parasitology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases National Institutes of Health  Bethesda Maryland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vinod","family":"Nair","sequence":"additional","affiliation":[{"name":"Microscopy Unit, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases National Institutes of Health  Hamilton Montana"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sundar","family":"Ganesan","sequence":"additional","affiliation":[{"name":"Biological Imaging Section, Research Technologies Branch, National Institute of Allergy and Infectious Diseases National Institutes of Health  Bethesda Maryland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michael 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