Enhancement of Transgene Expression by Mild Hypothermia Is Promoter Dependent in HEK293 Cells

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 alpha, 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 degrees C versus 37 degrees 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 degrees 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 degrees C. Taken together, this work demonstrates promoter-dependent hypothermia responses in HEK293 cells and emphasizes interactions between endogenous TFs and promoter sequences.

Automated summary

This study investigated the responses of different exogenous promoters to mild hypothermia in HEK293 cells, finding that CMV promoter showed significant enhancement of EGFP expression at low temperature. The study also revealed the importance of the CMV enhancer region and the impact of NKX3-1 on EGFP expression.