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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We have investigated the DNA methylation patterns in genomically imprinted genes of the mouse. Both Igf2 and H19 are associated with clear‐cut regions of allele‐specific paternal modification in late embryonic and adult tissues. By using a sensitive PCR assay, it was possible to follow the methylation state of individual HpaII sites in these genes through gametogenesis and embryogenesis. Most of these CpG moieties are not differentially modified in the mature gametes and also become totally demethylated in the early embryo in a manner similar to non‐imprinted endogenous genes. Thus, the overall allele‐specific methylation pattern at these sites must be established later during embryogenesis after the blastula stage. In contrast, sites in an Igf2r gene intron and one CpG residue in the Igf2 upstream region have allele‐specific modification patterns which are established either in the gametes or shortly after fertilization and are preserved throughout pre‐implantation embryogenesis. These studies suggest that only a few DNA modifications at selective positions in imprinted genes may be candidates for playing a role in the maintenance of parental identity during development.

The capacity to efficiently transduce nondividing cells, shuttle large genetic payloads, and maintain stable long-term transgene expression are attributes that have brought lentiviral vectors to the forefront of gene delivery vehicles for research and therapeutic applications in a clinical setting. Our discussion initiates with advances in lentiviral vector development and how these sophisticated lentiviral vectors reflect improvements in safety, regarding the prevention of replication competent lentiviruses (RCLs), vector mobilization, and insertional mutagenesis. Additionally, we describe conventional molecular regulatory systems to manage gene expression levels in a spatial and temporal fashion in the context of a lentiviral vector. State of the art technology for lentiviral vector production by transient transfection and packaging cell lines are explicitly presented with current practices used for concentration, purification, titering, and determining the safety of a vector stock. We summarize lentiviral vector applications that have received a great deal of attention in recent years including the generation of transgenic animals and the stable delivery of RNA interference molecules. Concluding remarks address some of the successes in preclinical animals, and the recent transition of lentiviral vectors to human clinical trials as therapy for a variety of infectious and genetic diseases.

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