Abstract
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, EF1α, 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 °C versus 37 °C 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 °C, 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 °C. Taken together, this work demonstrates promoter-dependent hypothermia responses in HEK293 cells and emphasizes interactions between endogenous TFs and promoter sequences.
Funder
National Research Foundation of Korea
Subject
Paleontology,Space and Planetary Science,General Biochemistry, Genetics and Molecular Biology,Ecology, Evolution, Behavior and Systematics
Cited by
4 articles.
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