{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,28]],"date-time":"2025-10-28T15:11:25Z","timestamp":1761664285773,"version":"build-2065373602"},"reference-count":22,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2021,8,30]],"date-time":"2021-08-30T00:00:00Z","timestamp":1630281600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003725","name":"National Research Foundation of Korea","doi-asserted-by":"publisher","award":["2021R1A2C4002733, 2019R1A6A1A11051471"],"award-info":[{"award-number":["2021R1A2C4002733, 2019R1A6A1A11051471"]}],"id":[{"id":"10.13039\/501100003725","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Life"],"abstract":"<jats:p>Mild hypothermia has been widely used to enhance transgene expression and improve the cellular productivity of mammalian cells. This study investigated mild hypothermia-responsive exogenous promoters in human embryonic kidney 293 (HEK293) cells using site-specific integration of various promoter sequences, including CMV, EF1\u03b1, SV40, and TK promoters, into the well-known genomic safe harbor site, AAVS1. EGFP expression driven by the CMV promoter increased up to 1.5-fold at 32 \u00b0C versus 37 \u00b0C under stable expression, while others showed no hypothermic response. Integration of short CMV variants revealed that the CMV-enhancer region is responsible for the positive hypothermic response. CMV-enhancer-specific transcription factors (TFs) were then predicted through in silico analysis and RNA-sequencing analysis, resulting in the selection of one TF, NKX3-1. At 37 \u00b0C, overexpression of NKX3-1 in recombinant HEK293 cells expressing EGFP through the CMV promoter (CMV-EGFP) increased EGFP expression up to 1.6-fold, compared with that in CMV-EGFP, the expression level of which was comparable to that of CMV-EGFP at 32 \u00b0C. Taken together, this work demonstrates promoter-dependent hypothermia responses in HEK293 cells and emphasizes interactions between endogenous TFs and promoter sequences.<\/jats:p>","DOI":"10.3390\/life11090901","type":"journal-article","created":{"date-parts":[[2021,8,30]],"date-time":"2021-08-30T22:49:51Z","timestamp":1630363791000},"page":"901","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Enhancement of Transgene Expression by Mild Hypothermia Is Promoter Dependent in HEK293 Cells"],"prefix":"10.3390","volume":"11","author":[{"given":"Min Ho","family":"Jang","sequence":"first","affiliation":[{"name":"Department of Molecular Science and Technology, Ajou University, Suwon 16499, Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Honggi","family":"Min","sequence":"additional","affiliation":[{"name":"Department of Molecular Science and Technology, Ajou University, Suwon 16499, Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3291-2093","authenticated-orcid":false,"given":"Jae Seong","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Molecular Science and Technology, Ajou University, Suwon 16499, Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,8,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1110","DOI":"10.3109\/07388551.2015.1084266","article-title":"Human cell lines for biopharmaceutical manufacturing: History, status, and future perspectives","volume":"36","author":"Dumont","year":"2016","journal-title":"Crit. 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