In order to establish cells and organisms with predictable properties, synthetic biology makes use of controllable, synthetic genetic devices. These devices are used to replace or to interfere with natural pathways. Alternatively, they may be interlinked with endogenous pathways to create artificial networks of higher complexity. While these approaches have been already successful in prokaryotes and lower eukaryotes, the implementation of such synthetic cassettes in mammalian systems and even animals is still a major obstacle. This is mainly due to the lack of methods that reliably and efficiently transduce synthetic modules without compromising their regulation properties.To pave the way for implementation of synthetic regulation modules in mammalian systems we utilized lentiviral transduction of synthetic modules. A synthetic positive feedback loop, based on the Tetracycline regulation system was implemented in a lentiviral vector system and stably integrated in mammalian cells. This gene regulation circuit yields a bimodal expression response. Based on experimental data a mathematical model based on stochasticity was developed which matched and described the experimental findings. Modelling predicted a hysteretic expression responsewhich was verified experimentally. Thereby supporting the idea that the system is driven by stochasticity.The results presented here highlight that the combination of three independent tools/methodologies facilitate the reliable installation of synthetic gene circuits with predictable expression characteristics in mammalian cells and organisms.

Abstract:In this study, an in situ application for identifying neodymium (Nd) enriched surface materials that uses multitemporal hyperspectral images is presented (HySpex sensor). Because of the narrow shape and shallow absorption depth of the neodymium absorption feature, a method was developed for enhancing and extracting the necessary information for neodymium from image spectra, even under illumination conditions that are not optimal. For this purpose, the two following approaches were developed: (1) reducing noise and analyzing changing illumination conditions by averaging multitemporal image scenes and (2) enhancing the depth of the desired absorption band by deconvolving every image spectrum with a Gaussian curve while the rest of the spectrum remains unchanged (Richardson-Lucy deconvolution). To evaluate these findings, nine field samples from the Fen complex in Norway were analyzed using handheld X-ray fluorescence devices and by conducting detailed laboratory-based geochemical rare earth element determinations. The result is a qualitative outcrop map that highlights zones that are enriched in neodymium. To reduce the influences of non-optimal illumination, particularly at the studied site, a OPEN ACCESSRemote Sens. 2015, 7 5161 minimum of seven single acquisitions is required. Sharpening the neodymium absorption band allows for robust mapping, even at the outer zones of enrichment. From the geochemical investigations, we found that iron oxides decrease the applicability of the method. However, iron-related absorption bands can be used as secondary indicators for sulfidic ore zones that are mainly enriched with rare earth elements. In summary, we found that hyperspectral spectroscopy is a noninvasive, fast and cost-saving method for determining neodymium at outcrop surfaces.

Currently, lentiviral vectors for research and gene therapy are produced from 293-T cells that are transiently transfected with plasmids encoding the vector and helper functions. However, transiently transfected vectors as well as the presence of SV40 virus large T-antigen (T-Ag) cause serious technical and safety considerations. We aimed to exploit single copy integration sites in the HEK293 genome supporting lentiviral vector production. We found that lentiviral vectors result in minimal infectious particle production from single copy integrants in HEK293. Moreover, once this cell line harbors single copy integrations of lentiviral vectors, its ability to transiently produce lentiviral vectors becomes strongly impaired. T-Ag has a dramatic effect on virus production. Low levels of constitutive T-Ag expression can overcome the production restriction imposed by integrated lentiviral vectors copies. Interestingly, T-Ag does not exert its role at the level of transcriptional activity of the vector; rather, it seems to impose an indirect effect on the cell thereby enabling lentiviral vector production. Altogether, our study highlights the restrictions for integrated lentiviral vectors that are relevant for the establishment of stable and safe producer cell lines.

The improvement of safety and titer of retroviral vectors produced in standard retroviral packaging cell lines is hampered because production relies on uncontrollable vector integration events. The influences of chromosomal surroundings make it difficult to dissect the performance of a specific vector from the chromosomal surroundings of the respective integration site. Taking advantage of a technology that relies on the use of packaging cell lines with predefined integration sites, we have systematically evaluated the performance of several retroviral vectors. In two previously established modular packaging cell lines (Flp293A and 293 FLEX) with single, defined chromosomal integration sites, retroviral vectors were integrated by means of Flp-mediated site-specific recombination. Vectors that are distinguished by different long terminal repeat promoters were introduced in either the sense or reverse orientation. The results show that the promoter, viral vector orientation, and integration site are the main determinants of the titer. Furthermore, we exploited the viral production systems to evaluate read-through activity. Read-through is thought to be caused by inefficient termination of vector transcription and is inherent to the nature of retroviral vectors. We assessed the frequency of transduction of sequences flanking the retroviral vectors from both integration sites. The approach presented here provides a platform for systematic design and evaluation of the efficiency and safety of retroviral vectors optimized for a given producer cell line.

Long-term, recombinant gene expression in mammalian cells depends on the nature of the transgene integration site and its inherent properties to modulate transcription (epigenetic effects). Here we describe a method by which high transgene expression is achieved and stabilized in extensively proliferating cultures. The method is based on strict co-expression of the transgene with an antitoxin in cells that express the respective toxin. Since the strength of antitoxin expression correlates with an advantage for cell growth, the cells with strong antitoxin expression are enriched over time in cultures of heterogeneous cells. This principle was applied to CHO cell lines that conditionally express the toxin kid and that are transduced to co-express the antitoxin kis together with different transgenes of interest. Cultivation of pools of transfectants that express the toxin steadily increase their transgene expression within several weeks to reach a maximum that is up to 120-fold over the initial status. In contrast, average transgene expression drops in the absence of toxin expression. Together, we show that cells conditionally expressing kid can be employed to create overexpressing cells by a simple coupling of kis to the transgene of interest, without further manipulation and in absence of selectable drugs.

Skin diseases are usually treated using topical formulations. Frequently, multiple applications per day are necessary, as up to 90% of the formulation (and thus of the active) are withdrawn from the skin by contact with the environment. During the development of topical formulations ex vivo permeation and penetration experiments are deployed to characterize the formulations. Still, these tests do not take into account the removal of formulations during the application period. To date, only few methods exist to probe the substantivity of dermal formulations. The aim of this investigation was to develop methods that simulate skin-to-skin or clothing-to-skin contact and enable the determination of the amount of formulation that is removed from the skin due to the contact. Three different types of formulations were used to validate the systems: a conventional semisolid cream, an oil-in-oil-emulsion, and a film forming formulation. The results showed that the substantivity decreased in the order: film forming formulation > semisolid cream > oil-in-oil-emulsion. A similar trend could be determined with both methods although the total amounts of withdrawn formulation differed. The developed methods can add to the knowledge about the formulation and can be used to develop formulations that exhibit higher substantivity.

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