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Biomedical subjects

K Sue O'Shea

Publications and source records attributed to K Sue O'Shea.

8 recordsLinked to original sources
PubMed2006

Cellular inhibitors of long interspersed element 1 and Alu retrotransposition.

Long interspersed element (LINE) 1 retrotransposons are major genomic parasites that represent approximately 17% of the human genome. The LINE-1 ORF2 protein is also responsible for the mobility of Alu elements, which constitute a further approximately 11% of genomic DNA. Representative members of each element class remain mobile, and deleterious retrotransposition events can induce spontaneous genetic diseases. Here, we demonstrate that APOBEC3A and APOBEC3B, two members of the APOBEC3 family of human innate antiretroviral resistance factors, can enter the nucleus, where LINE-1 and Alu reverse transcription occurs, and specifically inhibit both LINE-1 and Alu retrotransposition. These data suggest that the APOBEC3 protein family may have evolved, at least in part, to defend the integrity of the human genome against endogenous retrotransposons.

APOBEC Deaminases↗
PubMed2006

Gene silencing using RNA interference in embryonic stem cells.

Pluripotent embryonic stem (ES) cells are an important model system to examine gene expression and lineage segregation during differentiation. One powerful approach to target and inhibit gene expression, RNAi, has been applied to ES cells with the goal of teasing out the cascades of gene expression/repression that shape the early embryo. In this chapter, we describe the current understanding of the mechanisms of gene silencing by small hairpin RNAs, as well as controls and caveats to using this approach in ES cells. A consideration of synthetic vs plasmid-based RNAi vectors, design of targeting constructs, transfection of ES cells, and flow sorting of targeted cells is followed by methods for the analysis of phenotype and behavior of targeted cell populations using immunohistochemistry, reverse transcriptase polymerase chain reaction, Western blotting, and scanning electron microscopy.

Animals↗
PubMed2004

Self-renewal vs. differentiation of mouse embryonic stem cells.

Embryonic stem (ES) cells are typically derived from the inner cell mass of the preimplantation blastocyst and can both self-renew and differentiate into all the cells and tissues of the embryo. Because they are pluripotent, ES cells have been used extensively to analyze gene function in development via gene targeting. The embryonic stem cell is also an unsurpassed starting material to begin to understand a critical, largely inaccessible period of development. If their differentiation could be controlled, they would also be an important source of cells for transplantation to replace cells lost through disease or injury or to replace missing hormones or genes. Traditionally, ES cells have been differentiated in suspension culture as embryoid bodies, named because of their similarity to the early postimplantation-staged embryo. Unlike the pristine organization of the early embryo, differentiation in embryoid bodies appears to be largely unpatterned, although multiple cell types form. It has recently been possible to separate the desired cell types from differentiating ES cells in embryoid bodies by using cell-type-restricted promoters driving expression of either antibiotic resistance genes or fluorophores such as EGFP. In combination with growth factor exposure, highly differentiated cell types have successfully been derived from ES cells. Recent technological advances such as RNA interference to knock down gene expression in ES cells are also producing enriched populations of cells and elucidating gene function in early development.

Animals↗
PubMed2004

Embryonic stem cells expressing expanded CAG repeats undergo aberrant neuronal differentiation and have persistent Oct-4 and REST/NRSF expression.

Nine neurodegenerative disorders are caused by CAG/polyglutamine (polyQ) repeat expansions. The molecular mechanisms responsible for disease-specific neurodegeneration remain elusive. We developed an embryonic stem (ES) cell-based model to probe the role of polyQ tract expansion in neuronal degeneration. ES cells containing expanded CAG repeats in the hypoxanthine phosphoribosyltransferase (Hprt) gene develop features typical of CAG-mediated neuropathology, exhibit length-dependent decrease in survival, undergo aberrant neuronal differentiation as well as persistent Oct-4 and Repressor element-1 transcription factor/neuron restrictive silencer factor (REST/NRSF) expression. This novel model will allow analysis of the molecular pathogenesis of neuronal degeneration and can be used to rapidly screen therapeutic interventions for these fatal diseases.

Cell Differentiation↗
PubMed2003

Oct4 RNA interference induces trophectoderm differentiation in mouse embryonic stem cells.

We examined whether suppression of Oct4 via RNA interference (RNAi) would affect embryonic stem (ES) cell lineage choice. Cells were transfected with plasmids containing an independently expressed reporter gene and an RNA polymerase type III promoter to constitutively express small stem-loop RNA transcripts corresponding to Oct4 mRNA. Cells transfected with Oct4 RNAi constructs demonstrated reduced levels of Oct4 mRNA and exhibited characteristics of trophectodermal differentiation. These findings support the critical role of Oct4 in regulating stem cell identity and suggest that future experiments using RNAi in ES cells can elucidate the roles of other genes affecting lineage specification during differentiation.

Animals↗
PubMed2003

RNA inhibition of BMP-4 gene expression in postimplantation mouse embryos.

Short, hairpin RNA (shRNA) directed against bone morphogenetic protein 4 (Bmp-4) was delivered to early postimplantation staged mouse embryos via tail vein injection of pregnant dams. As early as 24 h postinjection, embryos expressed a DsRed marker and later exhibited defects of neural fold elevation and closure and of cardiac morphogenesis. Immunohistochemical analysis of sectioned embryos indicated that Bmp-4 protein was depleted and gene expression analysis indicated there was a reduction in Bmp-4 mRNA and an upregulation of the Bmp-4 antagonists, noggin and chordin, in embryos exposed to the shRNA, but not in control embryos. There was no change in the expression of Gata4, brachyury, or claudin6 in RNAi exposed embryos, indicating that RNA silencing was specific to Bmp-4 rather than producing widespread gene inhibition. Delivery of shRNA to embryos has the potential to specifically knockdown the expression of developmentally essential genes and to rescue gene mutations, significantly decreasing the time required to analyze the function(s) of individual genes in development.

Animals↗
PubMed2002

Noggin and chordin have distinct activities in promoting lineage commitment of mouse embryonic stem (ES) cells.

To examine the role of secreted signaling molecules and neurogenic genes in early development, we have developed a culture system for the controlled differentiation of mouse embryonic stem (ES) cells. In the current investigation, two of the earliest identified BMP antagonists/neural-inducing factors, noggin and chordin, were expressed in pluripotent mouse ES cells. Neurons were present as early as 24 h following transfection of ES cells with a pCS2/noggin expression plasmid, with differentiation peaking at 72 h. With neuronal differentiation, stem cell marker genes were down-regulated and neural determination genes expressed. Coculture experiments and exposure to noggin-conditioned medium produced similar neuronal differentiation of control ES cells, while addition of BMP-4 to noggin expressants strikingly inhibited neuronal differentiation. Transfection of ES cells with a pCS2/chordin expression vector or exposure to chordin-conditioned medium produced a more complex pattern of differentiation; ES cells formed neurons, mesenchymal cells as well as N-CAM-positive, nestin-positive neuroepithelial progenitors. These data suggest that, consistent with their different expression fields, noggin and chordin may play distinct roles in patterning the early mouse embryo.

Animals↗