Use of the viral 2A peptide for bicistronic expression in transgenic mice

<p>Abstract</p> <p>Background</p> <p>Transgenic animals are widely used in biomedical research and biotechnology. Multicistronic constructs, in which several proteins are encoded by a single messenger RNA, are commonly used in genetically engineered animals. This is currently done by using an internal ribosomal entry site to separate the different coding regions. 2A peptides result in the co-translational 'cleavage' of proteins and are an attractive alternative to the internal ribosomal entry site. They are more reliable than the internal ribosomal entry site and lead to expression of multiple cistrons at equimolar levels. They work in a wide variety of eukaryotic cells, but to date have not been demonstrated to function in transgenic mice in an inheritable manner.</p> <p>Results</p> <p>To test 2A function in transgenic mice and uncover any possible toxicity of widespread expression of the 2A peptide, we made a bicistronic reporter construct containing the coding sequence for a membrane localised red fluorescent protein (Myr-TdTomato) and a nuclear localised green fluorescent protein (H2B-GFP), separated by a 2A sequence. When this reporter is transfected into HeLa cells, the two fluorescent proteins correctly localise to mutually exclusive cellular compartments, demonstrating that the bicistronic construct is a reliable readout of 2A function. The two fluorescent proteins also correctly localise when the reporter is electroporated into chick neural tube cells. We made two independent transgenic mouse lines that express the bicistronic reporter ubiquitously. For both lines, transgenic mice are born in Mendelian frequencies and are found to be healthy and fertile. Myr-TdTomato and H2B-GFP segregate to mutually exclusive cellular compartments in all tissues examined from a broad range of developmental stages, ranging from embryo to adult. One transgenic line shows X-linked inheritance of the transgene and mosaic expression in females but uniform expression in males, indicating that the transgene has integrated into the X chromosome in this line.</p> <p>Conclusion</p> <p>The 2A peptide efficiently mediates co-translational cleavage in transgenic mice in which it has been inherited through the germ-line. Mice expressing it ubiquitously throughout development and into adulthood appear normal. It is therefore a viable tool for use in genetically engineered mice and represents a superior alternative to the widely used internal ribosomal entry site.</p

The disparity of health facilities in an urban area discourages proposed treatment application in inoperable lung cancer patients

Georgios Hillas1, Petros Bakakos2, Miltiadis Trichas3, Fotis Vlastos11Department of Respiratory and Critical Care Medicine, &amp;ldquo;Sotiria&amp;rdquo; Chest Diseases Hospital, Athens, Greece; 21st Respiratory Medicine Department, University of Athens Medical School, &amp;ldquo;Sotiria&amp;rdquo; Chest Diseases Hospital, Athens, Greece; 3Radiotherapy Department, Metropolitan Hospital, Neo Faliro, GreeceObjectives: Patients with a newly diagnosed non-small cell lung cancer (NSCLC) stage IIIB are offered chemoradiotherapy, as proposed by the current guidelines. This combination treatment is facilitated by the coexistence of corresponding departments in the same establishment. The geographical disparity of these health facilities influences patients&amp;rsquo; willingness to be treated and may influence their survival. This is an observational study that compares the survival of two groups of patients with NSCLC stage IIIB: those treated with chemoradiotherapy versus those treated only with chemotherapy. These two comparable groups were formed exclusively by patients&amp;rsquo; and/or their families&amp;rsquo; decisions.Methods: One hundred fifteen consecutive NSCLC stage IIIB patients were included in the study. All were hospitalized in the biggest Chest Disease Hospital in Athens and were offered sequential chemoradiotherapy. Only 54 patients opted for the proposed treatment, while 61 decided to be treated with chemotherapy only, denying continuing their treatment in another health care unit (radiotherapy). Their survival and related factors were analyzed.Results: Mean overall survival was estimated 10 months (95% confidence interval [CI]: 7.96&amp;ndash;12.04). Patients treated with chemoradiotherapy had almost double overall survival compared to those under chemotherapy (P = 0.001): 13.6 months (95% CI: 12.3&amp;ndash;14.9) versus 7.5 (95% CI: 6.1&amp;ndash;8.9). Patients aged &amp;le; 65 years (P &amp;lt; 0.001), smokers (P &amp;lt; 0.001), and those without a cancer history (P &amp;lt; 0.001) survived longer.Conclusions: The lack of a radiotherapy department in a hospital providing chemotherapy impedes the application of current guidelines advocating combined radiochemotherapy. When recommended radiotherapy after six chemo cycles, half of the patients are unwilling to be displaced and do not follow the recommendations. This has an impact on patient survival.Keywords: non-small cell lung cancer, survival, radiotherapy, chemotherapy, health facilities&amp;nbsp

A Population of Dust-rich Quasars at z ~ 1.5

We report Herschel SPIRE (250, 350, and 500 μm) detections of 32 quasars with redshifts 0.5 ≤z < 3.6 from the Herschel Multi-tiered Extragalactic Survey (HerMES). These sources are from a MIPS 24 μm flux-limited sample of 326 quasars in the Lockman Hole Field. The extensive multi-wavelength data available in the field permit construction of the rest-frame spectral energy distributions (SEDs) from ultraviolet to the mid-infrared for all sources, and to the far-infrared (FIR) for the 32 objects. Most quasars with Herschel FIR detections show dust temperatures in the range of 25-60 K, with a mean of 34 K. The FIR luminosities range from 10^(11.3) to 10^(13.5) L_☉, qualifying most of their hosts as ultra- or hyper-luminous infrared galaxies. These FIR-detected quasars may represent a dust-rich population, but with lower redshifts and fainter luminosities than quasars observed at ~1 mm. However, their FIR properties cannot be predicted from shorter wavelengths (0.3-20 μm, rest frame), and the bolometric luminosities derived using the 5100 Å index may be underestimated for these FIR-detected quasars. Regardless of redshift, we observed a decline in the relative strength of FIR luminosities for quasars with higher near-infrared luminosities

Nodal dependent differential localisation of dishevelled-2 demarcates regions of differing cell behaviour in the visceral endoderm

The anterior visceral endoderm (AVE), a signalling centre within the simple epithelium of the visceral endoderm (VE), is required for anterior-posterior axis specification in the mouse embryo. AVE cells migrate directionally within the VE, thereby properly positioning the future anterior of the embryo and orientating the primary body axis. AVE cells consistently come to an abrupt stop at the border between the anterior epiblast and extra-embryonic ectoderm, which represents an end-point to their proximal migration. Little is known about the underlying basis for this barrier and how surrounding cells in the VE respond to or influence AVE migration. We use high-resolution 3D reconstructions of protein localisation patterns and time-lapse microscopy to show that AVE cells move by exchanging neighbours within an intact epithelium. Cell movement and mixing is restricted to the VE overlying the epiblast, characterised by the enrichment of Dishevelled-2 (Dvl2) to the lateral plasma membrane, a hallmark of Planar Cell Polarity (PCP) signalling. AVE cells halt upon reaching the adjoining region of VE overlying the extra-embryonic ectoderm, which displays reduced neighbour exchange and in which Dvl2 is excluded specifically from the plasma membrane. Though a single continuous sheet, these two regions of VE show distinct patterns of F-actin localisation, in cortical rings and an apical shroud, respectively. We genetically perturb PCP signalling and show that this disrupts the localisation pattern of Dvl2 and F-actin and the normal migration of AVE cells. In Nodal null embryos, membrane localisation of Dvl2 is reduced, while in mutants for the Nodal inhibitor Lefty1, Dvl2 is ectopically membrane localised, establishing a role for Nodal in modulating PCP signalling. These results show that the limits of AVE migration are determined by regional differences in cell behaviour and protein localisation within an otherwise apparently uniform VE. In addition to coordinating global cell movements across epithelia (such as during convergence extension), PCP signalling in interplay with TGFβ signalling can demarcate regions of differing behaviour within epithelia, thereby modulating the movement of cells within them

Elucidating the cellular basis for directional migration of anterior visceral endoderm in mouse embryo

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Μελέτη του φαινοτύπου των TAG-1 ελλειπτικών μυών και διερεύνηση του ρόλου της TAG-1 στη μετανάστευση των ολιγοδενδροκυττάρων

The development of the central neural system consists of complex cellular procedures including cell migration. The developing neurons migrate from their place of birth to their final destination in two directions: radially and tangentially. During the migration, the neural cells are guided by signal molecules which are proteins that act in an attractive or repulsive way. The combination of these proteins constitutes the guidance mechanism and defines the migration pattern the migrating neurons will follow. Like the neurons, the interactions between endogenous and exogenous signals induce the differentiation of the oligodendroglial lineage. In mouse, the oligodendrocyte precursors are born in the ventral part of the ventral neural tube at the age of Ε9.5, Ε10 and onwards, and migrate rostrally. This rostral migration could be affected by the above interactions.Η ανάπτυξη του Κεντρικού Νευρικού Συστήματος αποτελεί μια πολύπλοκη διαδικασία που περιλαμβάνει σημαντικές κυτταρικές λειτουργίες όπως κυτταρικός πολλαπλασιασμός, προγραμματισμένος κυτταρικός θάνατος κτλ. Θεμελιώδη θέση μεταξύ αυτών καταλαμβάνει η κυτταρική μετανάστευση. Τα νευρικά κύτταρα, στην πλειοψηφία τους, μεταναστεύουν μετα-μιτωτικά από την περιοχή γέννησης τους μέχρι τον τελικό προορισμό τους ο οποίος μπορεί να βρίσκεται σε μεγάλη απόσταση. Μελέτες πάνω στους μηχανισμούς μετανάστευσης έδειξαν την παρουσία δύο βασικών μεταναστευτικών τύπων: την ακτινωτή και την οριζόντια μετανάστευση. Κατά τη κίνηση τους, τα νευρικά κύτταρα ανταποκρίνονται σε διαφορετικά μόρια - σήματα του περιβάλλοντος τους που θα τα καθοδηγήσουν στις σωστές τους θέσεις. Τα μόρια-σήματα είναι πρωτεΐνες οι οποίες δρουν εξ επαφής η εξ αποστάσεως κατά ένα ελκτικό ή απωθητικό τρόπο. Η συνδυαστική δράση των συγκεκριμένων πρωτεϊνών συγκροτεί τον καθοδηγητικό μηχανισμό και καθορίζει το μεταναστευτικό πρότυπο που θα ακολουθήσουν τα μεταναστεύοντα κύτταρα. Όπως και στην περίπτωση των νευρώνων, αλληλεπιδράσεις μεταξύ ενδογενών κυτταρικών και τοπο-ειδικών εξωγενών σημάτων επάγουν και την αρχική διαφοροποίηση της ολιγοδενδρογλοιακής σειράς Στο μυ, τα πρόδρομα ολιγοδενδροκύτταρα γεννιούνται στο κοιλιακό τμήμα του νευρικού σωλήνα από την ηλικία Ε9.5, Ε10 και μετά, από όπου και μεταναστεύουν πρόσθια. Αυτή η πρόσθια μετανάστευση των πρόδρομων ολιγοδενδροκυττάρων προς τον τελικό τους προορισμό μπορεί να επηρεάζεται τις παραπάνω αλληλεπιδράσεις

Quantitative characterisation of rosettes.

<p>(A) Rosette density (number of rosettes divided by total VE cell number) at different wild-type stages (“pre-AVE”: before AVE induction, <i>n</i> = 9; “distal”: AVE at distal tip before migration, <i>n</i> = 5; “migrating”: AVE migrating, <i>n</i> = 5; and “anterior”: AVE finished proximal migration and moving laterally, <i>n</i> = 4) and in the AVE arrest mutants <i>Nodal^Δ600/lacZ</i> (<i>n</i> = 5) and <i>Cripto</i>^−/− (<i>n</i> = 9). There is a significant increase in rosettes' density in “migrating” embryos as compared to “distal” embryos. The AVE arrest mutants <i>Nodal^Δ600/lacZ</i> and <i>Cripto</i>^−/− show significantly reduced rosette density compared to “migrating” and “anterior” embryos, suggestive of a direct link between rosettes and AVE migration. (A′) The same data as in (A), but depicted as mean number of rosettes per embryo (blue line), and mean number of VE cells per embryo (green bars) at the various stages. “Migrating” embryos have a comparable number of VE cells to “distal” embryos, but have significantly more rosettes, leading to an increase in rosette density. AVE arrest mutants have similar average VE cell numbers to stage matched “anterior” embryos, but show significantly fewer rosettes, leading to the reduced rosette density. (B) Polar plot showing distribution of rosettes in the VE of embryos. Migrating AVE cells were used to determine the anterior of embryos. Rosettes are localised predominantly to the Epi-VE. Within the Epi-VE, rosettes appear to be uniformly distributed with respect to the anterior-posterior axis (<i>n</i> = 39 rosettes from 7 embryos). <i>p</i> values shown on the graphs were determined using Student's <i>t</i> test.</p

The VE contains multi-cellular rosettes.

<p>(A) A ZO-1 stained embryo in which cells are coloured in to illustrate the presence of junctions where three, four, or five cells meet at a point. (B) Rosettes are formed by five or more cells meeting at a point. A variety of rosettes are shown, including two that share some cells (last panel).</p