Assessing the efficiency of novel gene trap vectors in murine embryonic stem cells

Gene trapping is a random insertional mutagenesis strategy that aims to identify novel genes and analyse their function. It usually involves the introduction into embryonic stem (ES) cells of promoterless reporter/selector gene constructs whose expression can be activated only after integration downstream of a gene's regulatory elements. Gene trap insertions result in production of fusion transcripts consisting of the reporter and endogenous sequences and the mutated genes can be readily identified using PCR-based methods such as RACE. Furthermore the biological consequences of the integration event can be assessed after germ-line transmission. One limitation of conventional gene trapping is that it can only target genes expressed in ES cells since selection of insertional events relies on the endogenous promoter's activity to drive expression of the selectable marker and to circumvent this problem a new class of gene trap vectors called poly(A) trap vectors was developed. These constructs contain a 3' selectable marker whose expression is driven by a constitutively active internal promoter relaxing the requirement for endogenous gene expression. The selectable marker lacks a polyA signal but incorporates a splice donor (SD) signal so only integrations upstream of an endogenous gene's splice acceptor (SA) and polyA sequences can be selected thus eliminating intergenic background insertions. However, it has been recently demonstrated that poly(A) trap vectors are biased towards integrations into the 3'most-intron of their target genes due to the action of an mRNA-surveillance mechanism called nonsense-mediated mRNA decay (NMD).The aim of the study presented here was to assess the efficiency of a series of gene trap vectors that incorporate two novel features in their design: (i) the presence of an ATG-less, 5' triple fusion between egfp, P-galactosidase and neomycin/hygromycin resistance genes to function as a reporter/selector of the trapped gene's expression state and (ii) a 3' poly(A) trap cassette that contains the previously uncharacterized rabbit (3-globin exon 2/intron 2 SD junction and an AU-rich element (ARE) derived from the human GM-CSF gene. Our results provide evidence that the triple fusion functions properly and can be potentially used as a reporter of trapped locus activity. We also show that the presence of the ARE appears to improve the performance of the rabbit (3-globin SD sequence in the context of poly(A) trapping. More importantly, preliminary data suggest that our vectors may be resistant to NMD and thus potentially unbiased in their insertional preference

Understanding axial progenitor biology in vivo and in vitro

The generation of the components that make up the embryonic body axis, such as the spinal cord and vertebral column, takes place in an anterior-to-posterior (head-to-tail) direction. This process is driven by the coordinated production of various cell types from a pool of posteriorly-located axial progenitors. Here, we review the key features of this process and the biology of axial progenitors, including neuromesodermal progenitors, the common precursors of the spinal cord and trunk musculature. We discuss recent developments in the in vitro production of axial progenitors and their potential implications in disease modelling and regenerative medicine

In Vivo Differentiation Potential of Epiblast Stem Cells Revealed by Chimeric Embryo Formation

SummaryChimera formation after blastocyst injection or morula aggregation is the principal functional assay of the developmental potential of mouse embryonic stem cells (ESCs). This property, which demonstrates functional equivalence between ESCs and the preimplantation epiblast, is not shared by epiblast stem cell (EpiSC) lines. Here, we show that EpiSCs derived either from postimplantation embryos or from ESCs in vitro readily generate chimeras when grafted to postimplantation embryos in whole embryo culture. EpiSC derivatives integrate and differentiate to derivatives of all three embryonic germ layers and primordial germ cells. In contrast, grafted ESCs seldom proliferate in postimplantation embryos, and fail to acquire the identity of their host-derived neighbors. EpiSCs do not incorporate efficiently into embryonic day 8.5 embryos, a stage by which pluripotency has been lost. Thus, chimera formation by EpiSCs requires a permissive environment, the postimplantation epiblast, and demonstrates functional equivalence between this cell type and EpiSCs

MINERALOGICAL AND SPECTROSCOPIC STUDY OF NESQUEHONITE SYNTHESIZED BY REACTION OF GASEOUS CO2 WITH MG CHLORIDE SOLUTION

Στην παρούσα εργασία πραγματοποιήθηκε η σύνθεση νεσκεχονίτη, ενός ένυδρου ανθρακικού ορυκτού, υπό χαμηλές συνθήκες πίεσης με αντίδραση CO2 σε διάλυμα χλωριούχου μαγνησίου. Ο νεσκεχονίτης μπορεί να αξιοποιηθεί ως πρώτη ύλη σε δομικά υλικά και επιπλέον στην διαχείριση υγρών αποβλήτων. Ο νεσκεχονίτης μελετήθηκε με περιθλασιομετρία ακτίνων-Χ, υπέρυθρη φασματοσκοπία (FT-IR) και φασματοσκοπία Raman, διοφθάλμιο στερεοσκόπιο, Ηλεκτρονικό Μικροσκόπιο Σάρωσης και Ηλεκτρονικό Μικροσκόπιο Διερχόμενης Δέσμης Ηλεκτρονίων. Ο παραγόμενος νεσκεχονίτης αναπτύσσει επιμήκεις διαφανείς έως ημιδιαφανείς βελονοειδείς κρυστάλλους με υαλώδη λάμψη. Η υπέρυθρη φασματοσκοπία (FT-ΙR) και η φασματοσκοπία Raman υπέδειξαν την παρουσία ΟΗ- και HCO3 - στην κρυσταλλική δομή του νεσκεχονίτη. Η διαδικασία σύνθεσης που περιγράφεται στην παρούσα εργασία μπορεί να χρησιμοποιηθεί στην διαδικασία της ορυκτοποίησης για μόνιμη αποθήκευση των εκπομπών CO2Nesquehonite, a hydrous carbonate with promising uses such as building raw material and treatment of wastewaters, was synthesized under low pressure conditions by reaction of gaseous CO2 with Mg chloride solution and it was studied by means of X-Ray Diffraction, optical and scanning/transmission electron microscopy, and FTIR and Raman spectroscopic methods. Synthesized nesquehonite forms elongated fibers, exhibiting transparent to translucent diaphaneity and vitreous luster. It is characterized by high crystallinity. IR and Raman spectroscopy indicated the presence of OHand HCO3 - in the crystal structure of nesquehonite. The nesquehonite synthesis described herein constitutes a potential permanent storage of CO2 emissions

Inhibition of human lung cancer cell proliferation and survival by wine

Compounds of plant origin and food components have attracted scientific attention for use as agents for cancer prevention and treatment. Wine contains polyphenols that were shown to have anti-cancer and other health benefits. The survival pathways of Akt and extracellular signal-regulated kinase (Erk), and the tumor suppressor p53 are key modulators of cancer cell growth and survival. In this study, we examined the effects of wine on proliferation and survival of human Non-small cell lung cancer (NSCLC) cells and its effects on signaling events.Compounds of plant origin and food components have attracted scientific attention for use as agents for cancer prevention and treatment. Wine contains polyphenols that were shown to have anti-cancer and other health benefits. The survival pathways of Akt and extracellular signal-regulated kinase (Erk), and the tumor suppressor p53 are key modulators of cancer cell growth and survival. In this study, we examined the effects of wine on proliferation and survival of human Non-small cell lung cancer (NSCLC) cells and its effects on signaling events

Synthetic sodalite doped with silver nanoparticles : characterization and mercury (II) removal from aqueous solutions

In this work, a novel silver nanoparticles-doped synthetic sodalitic composite was synthesized and characterized using advanced characterization methods, namely TEM-EDS, XRD, SEM, XRF, BET, zeta potential, and particle size analysis. The synthesized nanocomposite was used for the removal of Hg2+ from 10 ppm aqueous solutions of initial pH equal to 2. The results showed that the sodalitic nanocomposites removed up to 98.65% of Hg2+, which is ∼16% and 70% higher than the removal achieved by sodalite and parent coal fly ash, respectively. The findings revealed that the Hg2+ removal mechanism is a multifaceted mechanism that predominantly involves adsorption, precipitation and Hg-Ag amalgamation. The study of the anions effect (Cl−, NO3−, C2H3O2−, and SO42−) indicated that the Hg2+ uptake is comparatively higher when Cl− anions co-exist with Hg2+ in the solution

Synthetic coal fly ash-derived zeolites doped with silver nanoparticles for mercury (II) removal from water

Coal fly ash-derived zeolites have attracted considerable interest in the last decade due to their use in several environmental applications such as the removal of dyes and heavy metals from aqueous solutions. In this work, coal fly ash-derived zeolites and silver nanoparticles-impregnated zeolites (nanocomposites) were synthesized and characterized by TEM/EDX, SEM/EDX, XRD, XRF, porosimetry (BET), particle size analysis (PSA) and zeta potential measurements. The synthesized materials were used for the removal of Hg2+ from aqueous solutions. The results demonstrated that nanocomposites can remove 99% of Hg2+, up to 10% and 90% higher than the removal achieved by the zeolite and the parent fly ash, respectively. Leaching studies further demonstrated the superiority of the nanocomposite over the parent materials. The Hg2+ removal mechanism is complex, involving adsorption, surface precipitation and amalgamation

In vitro generation of posterior motor neurons from human pluripotent stem cells

The ability to generate spinal cord motor neurons from human pluripotent stem cells (hPSCs) is of great use for modelling motor neuron–based diseases and cell-replacement therapies. A key step in the design of hPSC differentiation strategies aiming to produce motor neurons involves induction of the appropriate anteroposterior (A-P) axial identity, an important factor influencing motor neuron subtype specification, functionality, and disease vulnerability. Most current protocols for induction of motor neurons from hPSCs produce predominantly cells of a mixed hindbrain/cervical axial identity marked by expression of Hox paralogous group (PG) members 1-5, but are inefficient in generating high numbers of more posterior thoracic/lumbosacral Hox PG(8-13)+ spinal cord motor neurons. Here, we describe a protocol for efficient generation of thoracic spinal cord cells and motor neurons from hPSCs. This step-wise protocol relies on the initial generation of a neuromesodermal-potent axial progenitor population, which is differentiated first to produce posterior ventral spinal cord progenitors and subsequently to produce posterior motor neurons exhibiting a predominantly thoracic axial identity