Use of the cytomegalovirus promoter for transient and stable transgene expression in mouse embryonic stem cells.

Embryonic stem (ES) cells are pluripotent cells derived from the epiblast of preimplantation embryos. These cells are emerging as a key model system for elucidating mechanisms involved in development and disease as well as having a unique potential as a source of unlimited somatic cells for transplantation therapies. ES cells can be easily manipulated at the DNA level, allowing both transient and stable expression of complementary DNA encoding transgenes of interest. The human cytomegalovirus (CMV) immediate-early enhancer and promoter is commonly used for transient expression of transgenes in ES cells. However, its use in the formation of stable cell lines is less common. We demonstrate an electroporator transformation technique that results in up to 90% transfection efficiency of CMV-encoding vectors in ES cells. Furthermore, we describe the design of vectors and cloning techniques that allow stable expression of transgenes under control of the CMV promoter and a fluorescent microscopy method for detecting protein expression in ES cells in situ.

Making sense of intimate partner violence in late life: comments from online news readers.

PURPOSE: The purpose of this study was to gain insight into public awareness of intimate partner violence (IPV) in late life by how individuals respond to incidents of IPV reported in the newspaper. DESIGN AND METHODS: Using grounded theory techniques, online news items covering 24 incidents of IPV in late life, and the reader comments posted to them were analyzed. The news items were examined for incident details, story framing, and reporting style. An open coding process (Charmaz, K. [2006]. Constructing grounded theory: A practical guide through qualitative analysis. Thousand Oaks, CA: Sage Publications.) was used to generate a comprehensive understanding of themes and patterns in the comments posted by readers. RESULTS: Few posters indicated that incidents were episodes of IPV. As many posters struggled to make sense of incidents, they attempted to remove guilt from the perpetrator by assigning blame elsewhere. Comments were influenced by personal assumptions and perspectives about IPV, relationships, and old age; reporting style of the news items; and comments posted by other posters. IMPLICATIONS: Altering public views of IPV in late life requires raising awareness through education, reframing the ways in which information is presented, and placing greater emphasis on the context of the violence. By engaging interactive news media, reporters, participatory journalists, and policymakers can enhance public recognition and understanding of IPV in late life.

Embryonic expression of murine 5T4 oncofoetal antigen is associated with morphogenetic events at implantation and in developing epithelia.

Overexpression of 5T4 oncofoetal antigen, an early marker of ES cell differentiation, in vitro increases cellular motility and decreases adhesion, properties relevant to development and cancer. Embryonic expression of m5T4 antigen is first detected on trophectoderm at implantation and is restricted to extra-embryonic tissues to embryonic day (E) 11.5. In the embryo, significant m5T4 expression is detected at E12.5 in hindbrain roofplate and in various epithelia derived from all germ layers. In keratin 14-expressing epithelia, there is a congruent 5T4 expression pattern with many of these cells being Ki-67 positive. In brain, expression is observed in roofplate, ependymal layers, choroid plexus, and subventricular zones of lateral ventricles at E14.5. By E17.5, expression is decreased in the subventricular zone with further restriction to choroid plexus in adult brain. Our data demonstrate a limited 5T4 expression profile during embryogenesis associated with actively cycling, undifferentiated epithelial progenitor cells that may contribute to their migration.

Significant variations in differentiation properties between independent mouse ES cell lines cultured under defined conditions.

Mouse embryonic stem (ES) cells are isolated from the inner cell mass (ICM)/epiblast of preimplantation embryos and are widely used in cell differentiation studies. We have previously observed differences in transcript and antigen expression following differentiation of ES cells lines in vitro. We have investigated this further by comparing the differentiation characteristics of five independently derived ES cell lines cultured and differentiated under defined conditions. Undifferentiated ES cell lines exhibited similar morphology and antigen/transcript marker expression. However, upon differentiation in monolayer culture by LIF withdrawal, only two of the lines expressed similar germ layer transcript profiles, and these were significantly altered compared to differentiation in serum-supplemented media. Neurofilament-68k was the only transcript marker common to all cell lines, however, induction of neuroectoderm lineages using 1 microM all-trans retinoic acid (RA) resulted in significant variations in cell number and morphology between the lines. Furthermore, neurons were only formed from clones of the two cell lines that exhibited similar transcript profiles, although the morphology was different between the two. We conclude that the independent ES cell lines in this study differ in their response to alterations in culture conditions in vitro, and the use of an appropriate cell line enables relatively homogeneous neuronal populations to be achieved in monolayer culture under defined conditions.