The long-term production of billions of spermatozoa relies on the regulated proliferation and differentiation of spermatogonial stem cells (SSCs). To date only a few factors are known to function in SSCs to provide this regulation. Octamer-4 (OCT4) plays a critical role in pluripotency and cell survival of embryonic stem cells and primordial germ cells; however, it is not known whether it plays a similar function in SSCs. Here, we show that OCT4 is required for SSC maintenance in culture and for colonization activity following cell transplantation, using lentiviral-mediated short hairpin RNA expression to knock down OCT4 in an in vitro model for SSCs ("germline stem" [GS] cells). Expression of promyelocytic leukemia zinc-finger (PLZF), a factor known to be required for SSC self-renewal, was not affected by OCT4 knockdown, suggesting that OCT4 does not function upstream of PLZF. In addition to developing a method to test specific gene function in GS cells, we demonstrate that retinoic acid (RA) triggers GS cells to shift to a differentiated, premeiotic state lacking OCT4 and PLZF expression and colonization activity. Our data support a model in which OCT4 and PLZF maintain SSCs in an undifferentiated state and RA triggers spermatogonial differentiation through the direct or indirect downregulation of OCT4 and PLZF. The current study has important implications for the future use of GS cells as an in vitro model for spermatogonial stem cell biology or as a source of embryonic stem-like cells.

An important cell fate decision in Bacillus subtilis is shown to be the result of a ‘molecular race' between competing differentiation programs. The programs controlling competence initiation and spore formation progress independently, and without cross-talk, before cell fate choice.

The rat has served as an excellent model for studies on animal physiology and as a model for human diseases such as diabetes and alcoholism; however, genetic studies have been limited because of the inability to knock out genes. Our goal was to produce heritable deficiencies in specific gene function in the rat using RNA interference to knock down gene expression in vivo. Lentiviralmediated transgenesis was used to produce rats expressing a short hairpin RNA targeting Dazl, a gene expressed in germ cells and required for fertility in mice [Ruggiu, M., Speed, R., Taggart, M., McKay, S. J., Kilanowski, F., Saunders, P., Dorin, J. & Cooke, H. J. (1997) Nature 389, 73-77]. Germ-line transmission of the transgene occurred, and its expression correlated with significant reductions in DAZL protein levels and male sterility, and the knockdown was stable over multiple generations (F 1-F3). This study demonstrates an efficient system by which directed reverse genetic analysis can now be performed in the rat.Dazl ͉ fertility ͉ RNA interference T he Norway rat was the first mammal to be domesticated for scientific purposes, and over the last 150 years it has proven an invaluable research model, in part because of its size, fecundity, and ability to learn new behaviors with relative ease (1). The generation of new rat genetic models for the study of human diseases has been possible using forward genetic methods such as random mutagenesis and conventional transgenesis or through the discovery of serendipitous spontaneous mutations (2). However, reverse genetic methods crucial for analyzing the function of specific genes of interest are severely limited in the rat. Our goal was to produce heritable and stable deficiencies in specific gene function in this animal model. Gene inactivation through homologous recombination in ES cells is still not possible in the rat because of the lack of pluripotent ES cell lines similar to those available from the mouse. Instead, we chose to test an RNA interference (RNAi) approach that relies on the generation of transgenic rats.RNAi is a reverse genetic technology that allows one to down-regulate gene expression by introducing into cells a short (Ϸ19-to 21-nt) double-stranded RNA that is complementary to a target gene (3). In combination with the recent expansion of available genome sequence information, RNAi has provided a powerful tool affecting many areas of biological research. Whereas methods are now well established for performing RNAi in vitro using short interfering RNA, the application of RNAi to knock down gene expression in vivo is far less common. In most cases RNAi is achieved in vivo only transiently and is targeted to a particular tissue. Stable RNAi is usually accomplished by genetic modification of cells such that they carry a piece of DNA that contains a ubiquitous promoter (e.g., the Pol III promoters U6 or H1) that drives expression of a short hairpin RNA (shRNA). The shRNA is then processed to short interfering RNA by cellular machinery. Recent studies have shown that gen...

The concept of universe is a broad concept with limited analysis within nursing research and practice. The universe consists of everything that exists. The universe is all physical matter, including all the stars, planets, galaxies, and everything that exists within them such as human beings and nature. The concept analysis of theuniverse utilized the Walker and Avant eight-step methodology. The aim of the concept analysis was to provide knowledge and understanding of universe connection and interconnection in its entirety to human beings and health care, especially in advanced practice nursing. Attributes of the concept of universe include human being, environment, and cognition. Antecedents of the concept were observation, introspection, and experience. Lastly, the consequences of universe were self-awareness, evolution, and support.