Tumor necrosis factor alpha (TNF-alpha) signaling gives rise to a number of events, including activation of transcription factor NF-kappaB and programmed cell death (apoptosis). Previous studies of TNF-alpha signaling have suggested that these two events occur independently. The sensitivity and kinetics of TNF-alpha-induced apoptosis are shown to be enhanced in a number of cell types expressing a dominant-negative IkappaBalpha (IkappaBalphaM). These findings suggest that a negative feedback mechanism results from TNF-alpha signaling in which NF-kappaB activation suppresses the signals for cell death.

Methylation patterns of specific genes have been studied by polymerase chain reaction and found to undergo dynamic changes in the germ line and early embryo. Some CpG sites are methylated in sperm DNA and unmodified in mature oocytes, indicating that the parental genomes have differential methylation profiles. These differences, however, are erased by a series of early embryonic demethylation and postblastula remodification events, which serve to reestablish the basic adult methylation pattern prior to organogenesis. During gametogenesis, all of these sites are unmethylated in primordial germ cells but eventually become remodified by 18.5 days postcoitum in both males and females. The final methylation profile of the mature germ cells is then formed by a multistep process of site-specific demethylation events. These results form a basis for the understanding of the biochemical mechanisms and role of DNA methylation in embryonic development.

Successful gene therapy approaches will require efficient gene delivery and sustained expression of the transgene in recipients. A variety of methods, ranging from direct DNA delivery to infection with recombinant viruses containing foreign genes, have been developed, but they all have some major limitations that restrict their utility. We have described a human lentiviral (HIV)-based vector that can transduce non-dividing cells in vitro and deliver genes in vivo. With this vector, expression of transgenes in the brain has been detected for more than six months--the longest period tested so far. Because lentiviral vectors are pseudotyped with vesicular stomatitis virus G glycoprotein (VSVG; ref. 8), they can transduce a broad range of tissues and cell types. We now describe the ability of lentiviral vectors to introduce genes directly into liver and muscle. Sustained expression of green fluorescent protein (GFP), used as a surrogate for therapeutic protein, can be observed for more than 22 weeks in the liver. Similar long-term expression (more than eight weeks) was observed in transduced muscle. In contrast, little or no GFP could be detected in liver or muscle transduced with the Moloney murine leukaemia virus (M-MLV), a prototypic retroviral based vector. At a minimum, 3-4% of the total liver tissue was transduced by a single injection of 1-3 x 10(7) infectious units (I.U.) of recombinant HIV vector. Furthermore, no inflammation of recruitment of lymphocytes could be detected at the site of injection. Animals previously transduced with a lentiviral vector can be efficiently re-infected with lentiviral vectors. Additionally, we show that the requirement for lentiviral accessory proteins to establish efficient transduction in vivo is tissue dependent.

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