Osmotic pressure modulates single cell cycle dynamics inducing reversible growth arrest and reactivation of human metastatic cells

Biophysical cues such as osmotic pressure modulate proliferation and growth arrest of bacteria, yeast cells and seeds. In tissues, osmotic regulation takes place through blood and lymphatic capillaries and, at a single cell level, water and osmoregulation play a critical role. However, the effect of osmotic pressure on single cell cycle dynamics remains poorly understood. Here, we investigate the effect of osmotic pressure on single cell cycle dynamics, nuclear growth, proliferation, migration and protein expression, by quantitative time-lapse imaging of single cells genetically modified with fluorescent ubiquitination-based cell cycle indicator 2 (FUCCI2). Single cell data reveals that under hyperosmotic stress, distinct cell subpopulations emerge with impaired nuclear growth, delayed or growth arrested cell cycle and reduced migration. This state is reversible for mild hyperosmotic stress, where cells return to regular cell cycle dynamics, proliferation and migration. Thus, osmotic pressure can modulate the reversible growth arrest and reactivation of human metastatic cells

Abrogated cryptic activation of lentiviral transfer vectors

Despite significant improvements in lentivirus (LV) vector-based gene therapy there are still several safety risks using LV vectors including the potential formation of replication-competent LV particles. To address this shortcoming, we constructed a novel and safer gene transfer system using modified SIN-based LV gene transfer vectors. Central to our approach is a conditional deletion of the Ψ packaging signal after integration in the target genome. Here we demonstrate that after transduction of target cells, conventional SIN-based LV transfer vectors can still be mobilized. However mobilization is rendered undetectable if transductions are followed by a Cre/loxP-mediated excision of Ψ. Thus conditional elimination of the packaging signal may represent another advance in increasing the safety of LV vectors for gene therapeutic treatment of chronic diseases

An Inducible and Reversible Mouse Genetic Rescue System

Inducible and reversible regulation of gene expression is a powerful approach for uncovering gene function. We have established a general method to efficiently produce reversible and inducible gene knockout and rescue in mice. In this system, which we named iKO, the target gene can be turned on and off at will by treating the mice with doxycycline. This method combines two genetically modified mouse lines: a) a KO line with a tetracycline-dependent transactivator replacing the endogenous target gene, and b) a line with a tetracycline-inducible cDNA of the target gene inserted into a tightly regulated (TIGRE) genomic locus, which provides for low basal expression and high inducibility. Such a locus occurs infrequently in the genome and we have developed a method to easily introduce genes into the TIGRE site of mouse embryonic stem (ES) cells by recombinase-mediated insertion. Both KO and TIGRE lines have been engineered for high-throughput, large-scale and cost-effective production of iKO mice. As a proof of concept, we have created iKO mice in the apolipoprotein E (ApoE) gene, which allows for sensitive and quantitative phenotypic analyses. The results demonstrated reversible switching of ApoE transcription, plasma cholesterol levels, and atherosclerosis progression and regression. The iKO system shows stringent regulation and is a versatile genetic system that can easily incorporate other techniques and adapt to a wide range of applications

Silencing and Un-silencing of Tetracycline-Controlled Genes in Neurons

To identify the underlying reason for the controversial performance of tetracycline (Tet)-controlled regulated gene expression in mammalian neurons, we investigated each of the three components that comprise the Tet inducible systems, namely tetracyclines as inducers, tetracycline-transactivator (tTA) and reverse tTA (rtTA), and tTA-responsive promoters (Ptets). We have discovered that stably integrated Ptet becomes functionally silenced in the majority of neurons when it is inactive during development. Ptet silencing can be avoided when it is either not integrated in the genome or stably-integrated with basal activity. Moreover, long-term, high transactivator levels in neurons can often overcome integration-induced Ptet gene silencing, possibly by inducing promoter accessibility

Transplantation of canine olfactory ensheathing cells producing chondroitinase ABC promotes chondroitin sulphate proteoglycan digestion and axonal sprouting following spinal cord injury

Olfactory ensheathing cell (OEC) transplantation is a promising strategy for treating spinal cord injury (SCI), as has been demonstrated in experimental SCI models and naturally occurring SCI in dogs. However, the presence of chondroitin sulphate proteoglycans within the extracellular matrix of the glial scar can inhibit efficient axonal repair and limit the therapeutic potential of OECs. Here we have used lentiviral vectors to genetically modify canine OECs to continuously deliver mammalian chondroitinase ABC at the lesion site in order to degrade the inhibitory chondroitin sulphate proteoglycans in a rodent model of spinal cord injury. We demonstrate that these chondroitinase producing canine OECs survived at 4 weeks following transplantation into the spinal cord lesion and effectively digested chondroitin sulphate proteoglycans at the site of injury. There was evidence of sprouting within the corticospinal tract rostral to the lesion and an increase in the number of corticospinal axons caudal to the lesion, suggestive of axonal regeneration. Our results indicate that delivery of the chondroitinase enzyme can be achieved with the genetically modified OECs to increase axon growth following SCI. The combination of these two promising approaches is a potential strategy for promoting neural regeneration following SCI in veterinary practice and human patients

Identification of Five Interacting Binaries in the Galactic Bulge Survey

We present optical light curves, spectroscopy, and classification of five X-ray sources in the Chandra Galactic Bulge Survey (CXOGBS J174009.1–284725 (CX5), CXOGBS J173935.7–272935 (CX18), CXOGBS J173946.9–271809 (CX28), CXOGBS J173729.1–292804 (CX37), CXOGBS J174607.6–261547 (CX561)). These objects were selected based on bright optical counterparts which were quickly found to have emission lines in their optical spectra. This paper presents an illustration of GBS optical follow-up, targeting emission line objects. Of these five objects, four exhibit photometric variability in the Sloan r' band. CX5 shows a tentative period of 2.1 hr and is clearly an intermediate polar (IP). CX28 and CX37 both exhibit flickering with no clear period. Both are also suggested to be IPs. CX18 was observed to undergo two dwarf nova outbursts. Finally, CX561 shows no detectable variability, although its characteristics would be consistent with either a quiescent low-mass X-ray binary or cataclysmic variable

Telomerase Efficiently Elongates Highly Transcribing Telomeres in Human Cancer Cells

RNA polymerase II transcribes the physical ends of linear eukaryotic chromosomes into a variety of long non-coding RNA molecules including telomeric repeat-containing RNA (TERRA). Since TERRA discovery, advances have been made in the characterization of TERRA biogenesis and regulation; on the contrary its associated functions remain elusive. Most of the biological roles so far proposed for TERRA are indeed based on in vitro experiments carried out using short TERRA-like RNA oligonucleotides. In particular, it has been suggested that TERRA inhibits telomerase activity. We have exploited two alternative cellular systems to test whether TERRA and/or telomere transcription influence telomerase-mediated telomere elongation in human cancer cells. In cells lacking the two DNA methyltransferases DNMT1 and DNMT3b, TERRA transcription and steady-state levels are greatly increased while telomerase is able to elongate telomeres normally. Similarly, telomerase can efficiently elongate transgenic inducible telomeres whose transcription has been experimentally augmented. Our data challenge the current hypothesis that TERRA functions as a general inhibitor of telomerase and suggest that telomere length homeostasis is maintained independently of TERRA and telomere transcription

Inducible Transgenic Rat Model for Diabetes Mellitus Based on shRNA-Mediated Gene Knockdown

The rat is an important animal model in biomedical research, but gene targeting technology is not established for this species. Therefore, we aimed to produce transgenic knockdown rats using shRNA technology and pronuclear microinjection. To this purpose, we employed a tetracycline-inducible shRNA expression system targeting the insulin receptor (IR). Doxycycline (DOX) treatment of the resulting transgenic rats led to a dose-dependent and reversible increase in blood glucose caused by ubiquitous inhibition of IR expression and signalling. We could neither detect an interferon response nor disturbances in microRNA processing after DOX treatment excluding toxic effects of shRNA expression. Low dose DOX treatment induced a chronic state of diabetes mellitus. In conclusion, we have developed a technology which allows the specific, inducible, and reversible suppression of any gene of interest in the rat. Our first transgenic rat line generated with this method represents an inducible model for diabetes mellitus

A novel piggybac transposon inducible expression system identifies a role for akt signalling in primordial germ cell migration

In this work, we describe a single piggyBac transposon system containing both a tet-activator and a doxycycline-inducible expression cassette. We demonstrate that a gene product can be conditionally expressed from the integrated transposon and a second gene can be simultaneously targeted by a short hairpin RNA contained within the transposon, both in vivo and in mammalian and avian cell lines. We applied this system to stably modify chicken primordial germ cell (PGC) lines in vitro and induce a reporter gene at specific developmental stages after injection of the transposon-modified germ cells into chicken embryos. We used this vector to express a constitutively-active AKT molecule during PGC migration to the forming gonad. We found that PGC migration was retarded and cells could not colonise the forming gonad. Correct levels of AKT activation are thus essential for germ cell migration during early embryonic development