Heterologous mammalian gene regulation systems for adjustable expression of multiple transgenes are necessary for advanced human gene therapy and tissue engineering, and for sophisticated in vivo gene-function analyses, drug discovery, and biopharmaceutical manufacturing. The antibiotic-dependent interaction between the repressor (E) and operator (ETR) derived from an Escherichia coli erythromycin-resistance regulon was used to design repressible (E(OFF)) and inducible (E(ON)) mammalian gene regulation systems (E.REX) responsive to clinically licensed macrolide antibiotics (erythromycin, clarithromycin, and roxithromycin). The E(OFF) system consists of a chimeric erythromycin-dependent transactivator (ET), constructed by fusing the prokaryotic repressor E to a eukaryotic transactivation domain that binds and activates transcription from ETR-containing synthetic eukaryotic promoters (P(ETR)). Addition of macrolide antibiotic results in repression of transgene expression. The E(ON) system is based on E binding to artificial ETR-derived operators cloned adjacent to constitutive promoters, resulting in repression of transgene expression. In the presence of macrolides, gene expression is induced. Control of transgene expression in primary cells, cell lines, and microencapsulated human cells transplanted into mice was demonstrated using the E.REX (E(OFF) and E(ON)) systems. The macrolide-responsive E.REX technology was functionally compatible with the streptogramin (PIP-regulated and tetracycline (TET-regulated expression systems, and therefore may be combined for multiregulated multigene therapeutic interventions in mammalian cells and tissues.

In multicellular systems cell identity is imprinted by epigenetic regulation circuits, which determine the global transcriptome of adult cells in a cell phenotype-specific manner. By combining two repressors, which control each other's expression, we have developed a mammalian epigenetic circuitry able to switch between two stable transgene expression states after transient administration of two alternate drugs. Engineered Chinese hamster ovary cells (CHO-K1) showed toggle switch-specific expression profiles of a human glycoprotein in culture, as well as after microencapsulation and implantation into mice. Switch dynamics and expression stability could be predicted with mathematical models. Epigenetic transgene control through toggle switches is an important tool for engineering artificial gene networks in mammalian cells.

Bistable and hysteretic switches, enabling cells to adopt multiple internal expression states in response to a single external input signal, have a pivotal impact on biological systems, ranging from cell-fate decisions to cell-cycle control. We have designed a synthetic hysteretic mammalian transcription network. A positive feedback loop, consisting of a transgene and transactivator (TA) cotranscribed by TA's cognate promoter, is repressed by constitutive expression of a macrolide-dependent transcriptional silencer, whose activity is modulated by the macrolide antibiotic erythromycin. The antibiotic concentration, at which a quasi-discontinuous switch of transgene expression occurs, depends on the history of the synthetic transcription circuitry. If the network components are imbalanced, a graded rather than a quasi-discontinuous signal integration takes place. These findings are consistent with a mathematical model. Synthetic gene networks, which are able to emulate natural gene expression behavior, may foster progress in future gene therapy and tissue engineering initiatives.synthetic biology ͉ synthetic gene networks ͉ bistability ͉ erythromycin ͉ feedback-loop

The architecture of gene regulatory networks is reminiscent of electronic circuits. Modular building blocks that respond in a logical way to one or several inputs are connected to perform a variety of complex tasks. Gene circuit engineers have pioneered the construction of artificial gene regulatory networks with the intention to pave the way for the construction of therapeutic gene circuits for next-generation gene therapy approaches. However, due to the lack of a critical amount of eukaryotic cell-compatible gene regulation systems, the field has so far been limited to prokaryotes. Recent development of several mammalian cell-compatible expression control systems laid the foundations for the assembly of transcription control modules that can respond to several inputs. Herein, three approaches to evoke combinatorial transcription control have been followed: (i) construction of artificial promoters with up to three operator sites for regulatory proteins, and (ii) parallel and (iii) serial linking of two gene regulation systems. We have combined tetracycline-, streptogramin-, macrolide-, and butyrolactone transcription control systems to engineer BioLogic gates of the NOT IF-, AND-, NOT IF IF-, NAND-, OR-, NOR-, and INVERTER-type in mammalian cells, which are able to respond to up to three different small molecule inputs. BioLogic gates enable logical transcriptional control in mammalian cells and, in combination with modern transduction technologies, could serve as versatile tools for regulated gene expression and as building blocks for complex artificial gene regulatory networks for applications in gene therapy, tissue engineering, and biotechnology. B