{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,14]],"date-time":"2026-04-14T06:11:58Z","timestamp":1776147118086,"version":"3.50.1"},"reference-count":47,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,4,14]],"date-time":"2026-04-14T00:00:00Z","timestamp":1776124800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Bioeng. Biotechnol."],"abstract":"<jats:sec>\n                    <jats:title>Introduction<\/jats:title>\n                    <jats:p>Mammalian transient gene expression technology is an essential tool for biotherapeutics engineering and production. Various reported transient expression systems in human embryonic kidney (HEK) 293 or Chinese hamster ovary (CHO) cells display different expression patterns and production yields, yet the mechanisms to which these patterns and yields can be attributed remain unknown.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      To identify the determining factors, we have dissected the transient expression processes through a three-snapshot approach for four commercially available transient production systems: Expi293\n                      <jats:sup>TM<\/jats:sup>\n                      PRO, Expi293F\u2122, ExpiCHO-S\u2122, and CHO4Tx\u00ae. A novel surface-display molecule fused with a CD4 transmembrane domain was designed to utilize flow cytometry to monitor transient expression stages of reaching cell surfaces prior to release into the extracellular medium. A cytosolic green fluorescence protein (GFP) was transfected as a readout for transfection efficiency, DNA transcription, and protein translation. The final stage of transient expression was monitored by expression titers for antibody proteins, which reflected the secretion and accumulation of protein products in culture media.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      The data showed that in response to titrated amounts of transfected DNAs for both the surface-display fusion molecule and the cytosolic GFP, Expi293\n                      <jats:sup>TM<\/jats:sup>\n                      PRO exhibited a unique sigmoidal-like curve, which was different from the logarithmic-like saturation curves for three other tested cell hosts. These results indicated that the Expi293\n                      <jats:sup>TM<\/jats:sup>\n                      PRO cells possessed a high sensitivity to subtle changes in the quantities of transfected DNAs at the stage of protein synthesis. The Expi293\n                      <jats:sup>TM<\/jats:sup>\n                      PRO cells were found to achieve very high transient expression, with more than 40 constructs attaining a titer of more than 1\u00a0g\/L, which could likely be credited to their unusual capability in utilizing transfected DNA for protein synthesis. Interestingly, when the plasmid DNAs encoding the target genes were replaced with the DNAs of empty expression vectors for transfection, a dramatic expression enhancement during the early production phase was observed for the transient Expi293\n                      <jats:sup>TM<\/jats:sup>\n                      PRO system.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>These results together suggested that the efficiency in handling transfected DNAs for protein synthesis (i.e., the ability to make intracellularly up-taken plasmid DNA complexes competent for DNA transcription in the nucleus) is likely a key limiting factor for effective transient production.<\/jats:p>\n                  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