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, 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.

Adaptation of STARR-seq method to be used with 3rd generation integrase-deficient promoterless lentiviral vectors

ABSTRACTAbility to functionally screen gene regulatory sequences, such as promoters and enhancers, in high throughput manner is an important prerequisite for many basic and translational research programs. One of the methods that allow such screening is STARR-seq, or self-transcribing active regulatory region sequencing. It allows to quickly screen millions of candidate sequences in the cell type of interest. However, it does rely on transfection as a delivery method which can be a limiting-step for some hard-to-transfect cells such as senescent cells. Here we show that integration-deficient and integration-competent promoterless lentiviral particles can be used to deliver STARR-seq constructs into cells. These constructs reported CMV enhancer activity both at protein and mRNA level. While further validations are necessary, ability to deliver STARR-seq libraries using lentiviral particles will significantly improve the versatility and usability of such a method.

Gene activation by a CRISPR-assisted trans enhancer

The deactivated CRISPR/Cas9 (dCas9) is now the most widely used gene activator. However, current dCas9-based gene activators are still limited by their unsatisfactory activity. In this study, we developed a new strategy, the CRISPR-assisted trans enhancer, for activating gene expression at high efficiency by combining dCas9-VP64/sgRNA with the widely used strong CMV enhancer. In this strategy, CMV enhancer DNA was recruited to target genes in trans by two systems: dCas9-VP64/csgRNA-sCMV and dCas9-VP64-GAL4/sgRNA-UAS-CMV. The former recruited trans enhancer by annealing between two short complementary oligonucleotides at the ends of the sgRNA and trans enhancer. The latter recruited trans enhancer by binding between GAL4 fused to dCas9 and UAS sequence of trans enhancer. The trans enhancer activated gene transcription as the natural looped cis enhancer. The trans enhancer could activate both exogenous reporter genes and variant endogenous genes in various cells, with much higher activation efficiency than that of current dCas9 activators.

Gene activation by a CRISPR-assistant trans enhancer

Gene activation is essential to the basic biological research and biomedicine. Therefore, various gene activators such as activation domain-ZNF, TALE and CRISPR proteins have been developed for this end, in which the CRISPR protein dead Cas9 (dCas9) is now most widely used. However, the current gene activators are still limited by their inefficient gene activation activity. In this study, we developed a new strategy, CRISPR-assistant trans enhancer, for activating gene expression in high efficiency by combining dCas9-VP64/sgRNA with a widely used strong enhancer, the CMV enhancer. In this strategy, a trans CMV enhancer DNA was recruited to target gene by dCas9-VP64/sgRNA via annealing between 3’ end of sgRNA and CMV enhancer. The trans enhancer activates gene transcription as the natural looped cis enhancer. The trans enhancer could activate both exogenous reporter gene and variant endogenous genes in various cells, with much higher activation efficiency than the current dCas9 activators.

Characterisation of a stably integrated expression system for exogenous protein expression in DT40

The use of constitutive promoters to drive exogenous protein expression is an important tool for the study of diverse biological processes. To create and characterise a stably integrated expression system for DT40 cells, we constructed integration cassettes for three commonly used promoter elements; CMV (cytomegalovirus), CBA (chicken beta actin) or CAG (a hybrid promoter containing the CMV enhancer and chicken beta actin promoter), and used these to stably integrate a TOPBP1 transgene at the OVA locus, a transcriptionally silent locus commonly used in DT40. We next performed a comparative analysis of protein expression levels and identified CAG as the most efficient of the promoter elements we have tested in DT40 cells. To assess whether the site of integration affected the levels of transgene expression, a second chromosomal locus, immediately adjacent to the endogenous TOPBP1 gene, was tested for CAG. No major differences in TopBP1 overexpression were observed. This confirms that use of the OVA locus for integrating transgenes is a rational choice for DT40. Finally, we demonstrate that our stably integrated overexpression system (SIOS) constructs can be efficiently excised by the induction of tamoxifen-regulated Cre expression. Taken together, SIOS is an easy-to-use and versatile system for constitutive, reversible exogenous protein production that provides a range of potential expression levels. This will be a useful tool for future DT40 experiments.