Abstract
The Sleeping Beauty (SB) transposon system is an efficient non-viral tool for gene transfer into a variety of cells, including human cells. Through a cut-and-paste mechanism, your favorite gene (YFG) is integrated into AT-rich regions within the genome, providing stable long-term 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-like particles (eVLPs) for vaccination. We provide step-by-step 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-CoV-2 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 µg to 5 mg). © Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Generation of the SB plasmids Basic Protocol 2: Generation of a stable HEK293T cell line constitutively secreting MLV-based eVLPs Basic Protocol 3: Evaluation of the SB constructs by immunofluorescence assay Basic Protocol 4: Validation of eVLPs by denaturing PAGE and western blot Alternate Protocol 1: Analysis of SARS-CoV-2 Spike protein oligomerization using blue native gel electrophoresis and western blot Alternate Protocol 2: Evaluation of eVLP quality by electron microscopy (negative staining) Basic Protocol 5: Small-scale production of eVLPs Alternate Protocol 3: Large-scale production of eVLPs (up to about 1 to 3 mg VLPs) Alternate Protocol 4: Large-scale production of eVLPs (up to about 3 to 5 mg VLPs) Support Protocol: Quantification of total protein concentration by Bradford assay.