Enzymatic engineering of the porcine genome with transposons and recombinases

<p>Abstract</p> <p>Background</p> <p>Swine is an important agricultural commodity and biomedical model. Manipulation of the pig genome provides opportunity to improve production efficiency, enhance disease resistance, and add value to swine products. Genetic engineering can also expand the utility of pigs for modeling human disease, developing clinical treatment methodologies, or donating tissues for xenotransplantation. Realizing the full potential of pig genetic engineering requires translation of the complete repertoire of genetic tools currently employed in smaller model organisms to practical use in pigs.</p> <p>Results</p> <p>Application of transposon and recombinase technologies for manipulation of the swine genome requires characterization of their activity in pig cells. We tested four transposon systems- <it>Sleeping Beauty</it>, <it>Tol2</it>, <it>piggyBac</it>, and <it>Passport </it>in cultured porcine cells. Transposons increased the efficiency of DNA integration up to 28-fold above background and provided for precise delivery of 1 to 15 transgenes per cell. Both Cre and Flp recombinase were functional in pig cells as measured by their ability to remove a positive-negative selection cassette from 16 independent clones and over 20 independent genomic locations. We also demonstrated a Cre-dependent genetic switch capable of eliminating an intervening positive-negative selection cassette and activating GFP expression from episomal and genome-resident transposons.</p> <p>Conclusion</p> <p>We have demonstrated for the first time that transposons and recombinases are capable of mobilizing DNA into and out of the porcine genome in a precise and efficient manner. This study provides the basis for developing transposon and recombinase based tools for genetic engineering of the swine genome.</p

Contributions of differential p53 expression in the spontaneous immortalization of a chicken embryo fibroblast cell line

BACKGROUND: The present study was carried out to determine whether the p53 pathway played a role in the spontaneous immortalization of the SC-2 chicken embryo fibroblast (CEF) cell line that has been in continuous culture for over three years. RESULTS: The SC-2 cell line emerged from an extended crisis period with a considerably slower growth rate than primary CEF cells. The phenotype of the SC-2 cells changed dramatically at about passage 80, appearing smaller than at earlier passages (e.g., passage 43) and possessing a small, compact morphology. This morphological change coincided with an increase in growth rate. Passage 43 SC-2 cells expressed undetectable levels of p53 mRNA, but by passage 95, the levels were elevated compared to primary passage 6 CEF cells and similar to levels in senescent CEF cells. However, the high level of p53 mRNA detected in passage 95 SC-2 cells did not correlate to functional protein activity. The expression levels of the p53-regulated p21(WAF1 )gene were significantly decreased in all SC-2 passages that were analyzed. Examination of the Rb pathway revealed that E2F-1 and p15(INK4b )expression fluctuated with increasing passages, with levels higher in passage 95 SC-2 cells compared to primary passage 6 CEF cells. CONCLUSION: The present study suggests that altered expression of genes involved in the p53 and Rb pathways, specifically, p53 and p21(WAF1), may have contributed to the immortalization of the SC-2 CEF cell line

The outer halos of elliptical galaxies

Recent progress is summarized on the determination of the density distributions of stars and dark matter, stellar kinematics, and stellar population properties, in the extended, low surface brightness halo regions of elliptical galaxies. With integral field absorption spectroscopy and with planetary nebulae as tracers, velocity dispersion and rotation profiles have been followed to ~4 and ~5-8 effective radii, respectively, and in M87 to the outer edge at ~150 kpc. The results are generally consistent with the known dichotomy of elliptical galaxy types, but some galaxies show more complex rotation profiles in their halos and there is a higher incidence of misalignments, indicating triaxiality. Dynamical models have shown a range of slopes for the total mass profiles, and that the inner dark matter densities in ellipticals are higher than in spiral galaxies, indicating earlier assembly redshifts. Analysis of the hot X-ray emitting gas in X-ray bright ellipticals and comparison with dynamical mass determinations indicates that non-thermal components to the pressure may be important in the inner ~10 kpc, and that the properties of these systems are closely related to their group environments. First results on the outer halo stellar population properties do not yet give a clear picture. In the halo of one bright galaxy, lower [alpha/Fe] abundances indicate longer star formation histories pointing towards late accretion of the halo. This is consistent with independent evidence for on-going accretion, and suggests a connection to the observed size evolution of elliptical galaxies with redshift.Comment: 8 pages. Invited review to appear in the proceedings of "Galaxies and their Masks" eds. Block, D.L., Freeman, K.C. & Puerari, I., 2010, Springer (New York

Mothers behaving badly: chaotic hedonism and the crisis of neoliberal social reproduction

This article focuses on the significance of the plethora of representations of mothers ‘behaving badly’ in contemporary anglophone media texts, including the films Bad Moms, Fun Mom Dinner and Bad Mom’s Christmas, the book and online cartoons Hurrah for Gin and the recent TV comedy dramas Motherland, The Let Down and Catastrophe. All these media texts include representations of, first, mothers in the midst of highly chaotic everyday spaces where any smooth routine of domesticity is conspicuous by its absence; and second, mothers behaving hedonistically, usually through drinking and partying, behaviour that is more conventionally associated with men or women without children. After identifying the social type of the mother behaving badly (MBB), the article locates and analyses it in relation to several different social and cultural contexts. These contexts are: a neoliberal crisis in social reproduction marked by inequality and overwork; the continual if contested role of women as ‘foundation parents’; and the negotiation of longer-term discourses of female hedonism. The title gestures towards a popular British sitcom of the 1990s, Men Behaving Badly, which popularized the idea of the ‘new lad’; and this article suggests that the new lad’s counterpart, the ladette, is mutating into the mother behaving badly, or the ‘lad mom’. Asking what work this figure does now, in a later neoliberal context, it argues that the mother behaving badly is simultaneously indicative of a widening and liberating range of maternal subject positions and symptomatic of a profound contemporary crisis in social reproduction. By focusing on the classed and racialised dynamics of the MBB – by examining who exactly is permitted to be hedonistic, and how – and by considering the MBB’s limited and partial imagining of progressive social change, the article concludes by emphasizing the urgency of creating more connections between such discourses and ‘parents behaving politically’

Ionized gas, molecules, and dust in Sh2-132

We analyze the various interstellar components of the HII region Sh2-132. The main stellar source is the double binary system that includes the Wolf-Rayet star WR153ab. We use radio continuum images at 408 and 1420 MHz, and HI 21cm line data taken from the Canadian Galactic Plane Survey, molecular observations of the 12CO(1-0) line at 115 GHz from the Five College Radio Astronomy Observatory, and available mid and far IR observations obtained with the MSX and IRAS satellites, respectively. Sh2-132 is composed of two shells showing radio continuum counterparts at both frequencies. The emission is thermal in nature. The estimated rms electron density and ionized mass of the nebula are n_e = 20 cm^{-3} and M_HII = 1500 Mo. The distribution of the CO emission shows molecular gas bordering the ionized nebula and interacting with it. The velocities of the molecular gas is in the range --38 to --53 km/s, similar to the velocity of the ionized gas. The emission at 8.3 mic. reveals a ring like feature of about 15' that encircles the bright optical regions. This emission is due to the PAHs and marks the location of photodissociation regions. The gas distribution in the environs of Sh2-132 can be explained in a scenario where the massive stars in the region photodissociated, ionized, and swept-up the dense molecular material from the parental cloud through their strong stellar winds and intense UV photon flux.Comment: 11 figures and 5 tables, accepted in MNRA

GASKAP -- The Galactic ASKAP Survey

A survey of the Milky Way disk and the Magellanic System at the wavelengths of the 21-cm atomic hydrogen (HI) line and three 18-cm lines of the OH molecule will be carried out with the Australian Square Kilometre Array Pathfinder telescope. The survey will study the distribution of HI emission and absorption with unprecedented angular and velocity resolution, as well as molecular line thermal emission, absorption, and maser lines. The area to be covered includes the Galactic plane (|b|< 10deg) at all declinations south of delta = +40deg, spanning longitudes 167deg through 360deg to 79deg at b=0deg, plus the entire area of the Magellanic Stream and Clouds, a total of 13,020 square degrees. The brightness temperature sensitivity will be very good, typically sigma_T ~ 1 K at resolution 30arcsec and 1 km/s. The survey has a wide spectrum of scientific goals, from studies of galaxy evolution to star formation, with particular contributions to understanding stellar wind kinematics, the thermal phases of the interstellar medium, the interaction between gas in the disk and halo, and the dynamical and thermal states of gas at various positions along the Magellanic Stream.Comment: 45 pages, 8 figures, Pub. Astron. Soc. Australia (in press

Hegemonic masculine conceptualisation in gang culture

This research sought to investigate the relationship between gang processes and differing forms of masculine expression. Three hundred and sixteen male participants, drawn from secondary schools within Cape Town, were included in the study. These schools were in areas differentially characterised by gang activity. The questionnaire included the newly devised Male Attitude Norm Inventory designed to explore hegemonic conceptualisations of masculinity. Factor analytic procedures rendered a three-factor model stressing the importance of male toughness, success and control. Through a series of t-tests for independent samples, as well as supporting qualitative data, participants from areas characterised by high gang activity were found to support these hegemonic elements to a significantly greater extent