{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,8]],"date-time":"2025-10-08T21:46:11Z","timestamp":1759959971864,"version":"3.41.2"},"reference-count":47,"publisher":"Oxford University Press (OUP)","issue":"8","license":[{"start":{"date-parts":[[2021,5,13]],"date-time":"2021-05-13T00:00:00Z","timestamp":1620864000000},"content-version":"vor","delay-in-days":1,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"name":"EU-funded projects","award":["270089"],"award-info":[{"award-number":["270089"]}]},{"name":"EDUFLUVAC","award":["FP7-HEALTH-2013-INNOVATION-1","602640"],"award-info":[{"award-number":["FP7-HEALTH-2013-INNOVATION-1","602640"]}]},{"name":"Portuguese \u201cFunda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia\u201d"},{"name":"R&D Projects","award":["PTDC\/EBB-EBI\/102266\/2008","EXPL\/BBB-BIO\/1541\/2013","IF\/01704\/2014\/CP1229\/CT0001","SFRH\/BD\/90564\/2012","SFRH\/BD\/86744\/2012"],"award-info":[{"award-number":["PTDC\/EBB-EBI\/102266\/2008","EXPL\/BBB-BIO\/1541\/2013","IF\/01704\/2014\/CP1229\/CT0001","SFRH\/BD\/90564\/2012","SFRH\/BD\/86744\/2012"]}]},{"name":"Investigador FCT","award":["IF\/01704\/2014"],"award-info":[{"award-number":["IF\/01704\/2014"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2021,8,7]]},"abstract":"<jats:title>Abstract<\/jats:title>\n               <jats:p>Insect Trichoplusia ni High Five\u2122 (Hi5) cells have been widely explored for production of heterologous proteins, traditionally mostly using the lytic baculovirus expression vector system (BEVS), and more recently using virus-free transient gene expression systems. Stable expression in such host cells would circumvent the drawbacks associated with both systems when it comes to scale-up and implementation of more efficient high-cell density process modes for the manufacturing of biologics. In this study, we combined Flipase (Flp) recombinase-mediated cassette exchange (RMCE) with fluorescence-activated cell sorting (FACS) for generating a stable master clonal Hi5 cell line with the flexibility to express single or multiple proteins of interest from a tagged genomic locus. The 3-step protocol herein implemented consisted of (i) introducing the RMCE docking cassette into the cell genome by random integration followed by selection in Hygromycin B and FACS (Hi5-tagging population), (ii) eliminating cells tagged in loci with low recombination efficiency by transfecting the tagging population with an eGFP-containing target cassette followed by selection in G418 and FACS (Hi5-RMCE population), and (iii) isolation of pure eGFP-expressing cells by FACS and expansion to suspension cultures (Hi5-RMCE master clone). Exchangeability of the locus in the master clone was demonstrated in small-scale suspension cultures by replacing the target cassette by one containing a single protein (i.e., iCherry, as an intracellular protein model) or two proteins (i.e., influenza HA and M1 for virus-like particles production, as an extracellular protein model). Overall, the stable insect Hi5 cell platform herein assembled has the potential to assist and accelerate biologics development.<\/jats:p>","DOI":"10.1093\/g3journal\/jkab166","type":"journal-article","created":{"date-parts":[[2021,5,13]],"date-time":"2021-05-13T04:38:00Z","timestamp":1620880680000},"source":"Crossref","is-referenced-by-count":7,"title":["Insect High Five\u2122 cell line development using site-specific flipase recombination technology"],"prefix":"10.1093","volume":"11","author":[{"given":"Mafalda M","family":"Dias","sequence":"first","affiliation":[{"name":"IBET, Instituto de Biologia Experimental e Tecnol\u00f3gica, Oeiras 2780-901, Portugal"},{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Oeiras 2780-901, Portugal"}]},{"given":"Jo\u00e3o","family":"Vidigal","sequence":"additional","affiliation":[{"name":"IBET, Instituto de Biologia Experimental e Tecnol\u00f3gica, Oeiras 2780-901, Portugal"},{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Oeiras 2780-901, Portugal"}]},{"given":"Daniela P","family":"Sequeira","sequence":"additional","affiliation":[{"name":"IBET, Instituto de Biologia Experimental e Tecnol\u00f3gica, Oeiras 2780-901, Portugal"},{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Oeiras 2780-901, Portugal"}]},{"given":"Paula M","family":"Alves","sequence":"additional","affiliation":[{"name":"IBET, Instituto de Biologia Experimental e Tecnol\u00f3gica, Oeiras 2780-901, Portugal"},{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Oeiras 2780-901, Portugal"}]},{"given":"Ana P","family":"Teixeira","sequence":"additional","affiliation":[{"name":"IBET, Instituto de Biologia Experimental e Tecnol\u00f3gica, Oeiras 2780-901, Portugal"},{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Oeiras 2780-901, Portugal"}]},{"given":"Ant\u00f3nio","family":"Rold\u00e3o","sequence":"additional","affiliation":[{"name":"IBET, Instituto de Biologia Experimental e Tecnol\u00f3gica, Oeiras 2780-901, Portugal"},{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Oeiras 2780-901, Portugal"}]}],"member":"286","published-online":{"date-parts":[[2021,5,12]]},"reference":[{"key":"2021100712032149500_jkab166-B1","doi-asserted-by":"crossref","first-page":"142","DOI":"10.1016\/S0958-1669(99)80024-X","article-title":"Modifying secretion and post-translational processing in insect cells","volume":"10","author":"Ailor","year":"1999","journal-title":"Curr Opin Biotechnol"},{"key":"2021100712032149500_jkab166-B2","doi-asserted-by":"crossref","first-page":"868","DOI":"10.1002\/jgm.1370","article-title":"Development of a scalable process for high-yield 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