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Human pluripotent stem cells (hPSCs) and their differentiated derivatives represent valuable tools for studying development, modeling diseases, and advancing cell therapy. Recent improvements in genome engineering allow for precise modifications of hPSCs, further enhancing their utility in basic and translational research. Here we describe a Recombinase-Mediated Cassette Exchange (RMCE) platform in hPSCs that allows for the highly efficient, rapid, and specific integration of transgenes. The RCME-mediated DNA integration process is nearly 100% efficient, without negatively affecting the pluripotency or karyotypic stability of hPSCs. Taking advantage of this convenient system, we first established a dual inducible expression system based on the Tet-On and Cumate-On systems, allowing for the inducible expression of two transgenes independently. Secondly, we incorporated a Tet-on inducible system, driving the expression of three genes simultaneously. However, two genes also contain independent degron sequences, allowing for precise control over the expression of each gene individually. We demonstrated the utility of these systems in hPSCs, as well as their functionality after differentiation into cells that were representative of the three germ layers. Lastly, we used the triple inducible system to investigate the lineage commitment induced by the pancreatic transcription factors NKX6.1 and PDX1. We found that controlled dual expression, but not individual expression, biases hPSC embryoid body differentiation towards the pancreatic lineage by inducing the expression of the NeuroD program. In sum, we describe a novel genetic engineering platform that allows for the efficient and fast integration of any desired transgene(s) in hPSCs using RMCE. We anticipate that the ability to modulate the expression of three transgenes simultaneously will further accelerate discoveries using stem cell technology. [ABSTRACT FROM AUTHOR]

The transgene toggling device is recognized as a powerful tool for gene- and cell-based biological research and precision medicine. However, many of these devices often operate in binary mode, exhibit unacceptable leakiness, suffer from transgene silencing, show cytotoxicity, and have low potency. Here, we present a novel transgene switch, SIQ, wherein all the elements for gene toggling are packed into a single vector. SIQ has superior potency in inducing transgene expression in response to tebufenozide compared with the Gal4/UAS system, while completely avoiding transgene leakiness. Additionally, the ease and versatility of SIQ make it possible with a single construct to perform transient transfection, establish stable cell lines by targeting a predetermined genomic locus, and simultaneously produce adenovirus for transduction into cells and mammalian tissues. Furthermore, we integrated a cumate switch into SIQ, called SIQmate, to operate a Boolean AND logic gate, enabling swift toggling-off of the transgene after the removal of chemical inducers, tebufenozide and cumate. Both SIQ and SIQmate offer precise transgene toggling, making them adjustable for various researches, including synthetic biology, genome engineering, and therapeutics.

Methylorubrum extorquens is an important model methylotroph and has enormous potential for the development of C1-based microbial cell factories. During strain construction, regulated promoters with a low background expression level are important genetic tools for expression of potentially toxic genes. Here we present an accordingly optimised promoter, which can be used for that purpose. During construction and testing of terpene production strains harbouring a recombinant mevalonate pathway, strong growth defects were observed which made strain development impossible. After isolation and characterisation of suppressor mutants, we discovered a variant of the cumate-inducible promoter PQ2148 used in this approach. Deletion of 28 nucleotides resulted in an extremely low background expression level, but also reduced the maximal expression strength to about 30% of the original promoter. This tightly repressed promoter version is a powerful module for controlled expression of potentially toxic genes in M. extorquens. [ABSTRACT FROM AUTHOR]

Cell-cell interactions underlie diverse physiological processes yet remain challenging to examine with conventional imaging tools. Here we report a novel strategy to illuminate cell proximity using transcriptional activators. We repurposed cumate, a small molecule inducer of gene expression, by caging its key carboxylate group with a nitrile. Nitrilase-expressing activator cells released the cage, liberating cumate for consumption by reporter cells. Reporter cells comprising a cumate-responsive switch expressed a target gene when in close proximity to the activator cells. Overall, this strategy provides a versatile platform to image and potentially manipulate cellular interactions over time.

More than half of licensed therapeutic recombinant proteins (r‐proteins) are manufactured using constitutively‐expressing, stably‐transfected Chinese hamster ovary (CHO) clones. While constitutive CHO expression systems have proven their efficacy for the manufacturing of monoclonal antibodies, many next‐generation therapeutics such as cytokines and bispecific antibodies as well as biological targets such as ectodomains of transmembrane receptors remain intrinsically challenging to produce. Herein, we exploited a cumate‐inducible CHO platform allowing reduced expression of various classes of r‐proteins during selection of stable pools. Following stable pool generation, fed‐batch productions showed that pools generated without cumate (OFF‐pools) were significantly more productive than pools selected in the presence of cumate (ON‐pools) for 8 out of the 10 r‐proteins tested, including cytokines, G‐protein coupled receptors (GPCRs), the HVEM membrane receptor ectodomain, the multifunctional protein High Mobility Group protein B1 (HMGB1), as well as monoclonal and bispecific T‐cell engager antibodies. We showed that OFF‐pools contain a significantly larger proportion of cells producing high levels of r‐proteins and that these cells tend to proliferate faster when expression is turned off, suggesting that r‐protein overexpression imposes a metabolic burden on the cells. Cell viability was lower and pool recovery was delayed during selection of ON‐pools (mimicking constitutive expression), suggesting that high producers were likely lost or overgrown by faster‐growing, low‐producing cells. We also observed a correlation between the expression levels of the GPCRs with Binding immunoglobulin Protein, an endoplasmic reticulum (ER) stress marker. Taken together, these data suggest that using an inducible system to minimize r‐protein expression during stable CHO pool selection reduces cellular stresses, including ER stress and metabolic burden, leading to pools with greater frequency of high‐expressing cells, resulting in improved volumetric productivity. [ABSTRACT FROM AUTHOR]

A novel inducible gene expression system using p-isopropyl benzoate (cumate) as an inducer was developed for the industrial production hosts, Bacillus subtilis and Bacillus megaterium. Cumate is non-toxic to the host, inexpensive, and carbon source-independent inducer which provides an economical option for large-scale production of valuable proteins and chemicals from Bacillus strains. The synthetic cumate-inducible system was constructed by combining the strong constitutive Bacillus promoter Pveg with regulatory elements of the Pseudomonas putida, CymR repressor, and its operator sequence CuO. The designed expression cassette containing a sfGFP reporter under the cumate-inducible promoter was assembled into a Bacillus-E. coli shuttle and gene expression investigated in the two Bacillus strains. Characterization of gene expression levels, expression kinetics, and dose-response to cumate inducer concentration confirmed high-level, but tightly controlled GFP reporter expression in tunable, cumate concentration-dependent manner. Unexpectedly, this expression system works equally well for Escherichia coli, resulting in a platform that can be used both in gram-positive and gram-negative expression host. Its tight regulation and controllable expression makes this system useful for metabolic engineering, synthetic biology studies as well industrial protein production. [ABSTRACT FROM AUTHOR]

Inducible gene expression systems are favored over stable expression systems in a wide variety of basic and applied research areas, including functional genomics, gene therapy, tissue engineering, biopharmaceutical protein production and drug discovery. This is because they are mostly reversible and thus more flexible to use. Furthermore, compared to constitutive expression, they generally exhibit a higher efficiency and have fewer side effects, such as cell death and delayed growth or development. Empowered by decades of development of inducible gene expression systems, researchers can now efficiently activate or suppress any gene, temporarily and quantitively at will, depending on experimental requirements and designs. Here, we review a number of most commonly used mammalian inducible expression systems and provide basic standards and criteria for the selection of the most suitable one.

In Trypanosoma brucei , the tetracycline-inducible system enables tightly-regulated, highly-efficient expression of recombinant proteins or double-stranded RNA in both procyclic and bloodstream form cells, providing useful molecular genetic tools to study gene functions. An alternative, vanillic acid-inducible system is recently described for procyclic T. brucei, providing ∼18-fold increase in GFP reporter expression upon induction (Sunter JD. Mol. Biochem. Parasitol. 2016, 207:45–48). Here we describe a cumate-inducible system that allows efficient, tunable gene expression showing >300-fold increase in GFP expression upon induction. The cumate-inducible system can be used alone or together with the tetracycline-inducible system, in both procyclic and bloodstream form T. brucei. Efficient cumate-inducible expression is also achieved in T. brucei -infected mice. [ABSTRACT FROM AUTHOR]

Ralstonia eutropha H16 ( Cupriavidus necator H16) is a Gram-negative, facultative chemolithoautotrophic bacterium which can use H 2 and CO 2 as sole energy and carbon sources in the absence of organic substrates. The biotechnological use of R. eutropha H16 on an industrial scale has already been established; however, only a small number of tools promoting inducible gene expression is available. Within this study two systems promoting inducible expression were designed on the basis of the strong j5 promoter and the Escherichia coli lacI or the Pseudomonas putida cumate regulatory elements. Both expression vectors display desired regulatory features and further increase the number of suitable inducible expression systems for the production of metabolites and proteins with R. eutropha H16. [ABSTRACT FROM AUTHOR]

Inducible gene expression systems are favored over stable expression systems in a wide variety of basic and applied research areas, including functional genomics, gene therapy, tissue engineering, biopharmaceutical protein production and drug discovery. This is because they are mostly reversible and thus more flexible to use. Furthermore, compared to constitutive expression, they generally exhibit a higher efficiency and have fewer side effects, such as cell death and delayed growth or development. Empowered by decades of development of inducible gene expression systems, researchers can now efficiently activate or suppress any gene, temporarily and quantitively at will, depending on experimental requirements and designs. Here, we review a number of most commonly used mammalian inducible expression systems and provide basic standards and criteria for the selection of the most suitable one. [ABSTRACT FROM AUTHOR]