Nuclear localization of the transcriptional activator NF-kappaB (nuclear factor kappaB) is controlled in mammalian cells by three isoforms of NF-kappaB inhibitor protein: IkappaBalpha, -beta, and - epsilon. Based on simplifying reductions of the IkappaB-NF-kappaB signaling module in knockout cell lines, we present a computational model that describes the temporal control of NF-kappaB activation by the coordinated degradation and synthesis of IkappaB proteins. The model demonstrates that IkappaBalpha is responsible for strong negative feedback that allows for a fast turn-off of the NF-kappaB response, whereas IkappaBbeta and - epsilon function to reduce the system's oscillatory potential and stabilize NF-kappaB responses during longer stimulations. Bimodal signal-processing characteristics with respect to stimulus duration are revealed by the model and are shown to generate specificity in gene expression.

The generation of high-titer, helper-free retroviruses by transient transection has been achieved by using the highy transfectable 293T cell line into which are stably introduced constructs that express retroviral packaging functions. The resulting ecotropic virus packing cell line BOSC 23 produces infectious retrovirus at >106 infectious units/ml of supernatant within 72 hr after CaPO4-mediated btansfection. A stringent assay for replication-competent virus showed that no helper virus was present. The system can produce high titers of retroviral vectors expressing genes that are extremely difficult to propagate at high titer in stable producer lines. This method should facilitate and extend the use of helper-free retroviral gene transfer, as weli as be useful for gene therapy. (B-galactosidase (3-gal) expression is directed by the viral promoter in the long terminal repeat and the neomycin resistance cassette is deleted], pCRIPenv-(6), pCRIPgag-2 (6), MFG-lacZ (7), MFG-tPA [similar to MFGlacZ, but expressing the human tissue plasminogen activator gene in place of (-gal (7)], pZAP (8), pSV2Hgm (9), pGPT2E (10), pGD (11), pGDv-abi (12), and pGD210bcr/abl (11).Enzymatic Assays and Nudeic Acid Prdures. Staining for (3-gal activity in intact cells and spleen was performed as described (13). Reverse transcriptase (RT) activity was assayed in the culture medium ofexponentially growing cells as described by Goff et al. (14). The in vitro abl kinase assays were performed as described by Konopka et al. (15) medium (17). The cells were then returned to the 37°C incubator (5% C02) for 24 hr. Subsequently, the medium was changed to 3 ml offresh 10%o FCS, and 24-48 hr later, the medium was removed and either filtered through a 0.45-,m filter or centrifuged at 500 x g for 5 min in a Sorvall RT6000B centrifuge. In experiments with chloroquine, the medium was changed to 10% FCS at 10 hr posttransfection and changed a second time at 24 hr posttransfection. Infections were performed as described (6). Viral titer was determined as the average number ofblue ((-gal-producing) cells per 10-25 high power fields (40,000-100,000 total cells) multiplied by a factor to account for magnification, plate size, and dilution of the infectious stock. When fluorescence-activated cell sorting (FACS) analysis was performed, the percentage of positive cells was multiplied by the total number of cells on the dish. G418 selection was performed as above except that at 48 hr postinfection the cells were split 1:10 into selective Abbreviations: (-gal, ,-galactosidase; G418R, G418 resistance; RT, reverse transcriptase; FCS, fetal calf serum; FACS, fluorescenceactivated cell sorting; SV40, simian virus 40; gpt, guanine phosphoribosyltransferase.

RNA interference (RNAi) has recently emerged as a specific and efficient method to silence gene expression in mammalian cells either by transfection of short interfering RNAs (siRNAs; ref. 1) or, more recently, by transcription of short hairpin RNAs (shRNAs) from expression vectors and retroviruses. But the resistance of important cell types to transduction by these approaches, both in vitro and in vivo, has limited the use of RNAi. Here we describe a lentiviral system for delivery of shRNAs into cycling and non-cycling mammalian cells, stem cells, zygotes and their differentiated progeny. We show that lentivirus-delivered shRNAs are capable of specific, highly stable and functional silencing of gene expression in a variety of cell types and also in transgenic mice. Our lentiviral vectors should permit rapid and efficient analysis of gene function in primary human and animal cells and tissues and generation of animals that show reduced expression of specific genes. They may also provide new approaches for gene therapy.

The B cell activating factor BAFF (BlyS/TALL-1/zTNF4) is a tumor necrosis factor (TNF)-related ligand that promotes B cell survival and binds to three receptors (BCMA, TACI, and the recently described BAFF-R). Here we report an absolute requirement for BAFF in normal B cell development. Examination of secondary lymphoid organs from BAFF-deficient mice revealed an almost complete loss of follicular and marginal zone B lymphocytes. In contrast, mice lacking BCMA had normal-appearing B lymphocyte compartments. BAFF therefore plays a crucial role in B cell development and can function through receptors other than BCMA.

B cell homeostasis has been shown to critically depend on BAFF, the B cell activation factor from the tumor necrosis factor (TNF) family. Although BAFF is already known to bind two receptors, BCMA and TACI, we have identified a third receptor for BAFF that we have termed BAFF-R. BAFF-R binding appears to be highly specific for BAFF, suggesting a unique role for this ligand-receptor interaction. Consistent with this, the BAFF-R locus is disrupted in A/WySnJ mice, which display a B cell phenotype qualitatively similar to that of the BAFF-deficient mice. Thus, BAFF-R appears to be the principal receptor for BAFF-mediated mature B cell survival.

SummaryNotch is a highly conserved transmembrane protein that is involved in cell fate decisions and is found in organisms ranging from Drosophila to humans. A human homologue of Notch, TAN1, was initially identified at the chromosomal breakpoint of a subset of T-cell lymphoblastic leukemias/lymphomas containing a t(7;9) chromosomal translocation; however, its role in oncogenesis has been unclear. Using a bone marrow reconstitution assay with cells containing retrovirally transduced TAN1 alleles, we analyzed the oncogenic potential of both nuclear and extranuclear forms of truncated TAN1 in hematopoietic cells. Although the Moloney leukemia virus long terminal repeat drives expression in most hematopoietic cell types, retroviruses encoding either form of the TAN1 protein induced clonal leukemias of exclusively immature T cell phenotypes in "~ of transplanted animals. All tumors overexpressed truncated TAN1 of the size and subcellular localization predicted from the structure of the gene. These results show that TAN1 is an oncoprotein and suggest that truncation and overexpression are important determinants of transforming activity. Moreover, the murine tumors caused by TAN1 in the bone marrow transplant model are very similar to the TANl-associated human tumors and suggest that TAN1 may be specifically oncotropic for T cells.

The control of myelination by oligodendrocytes in the CNS is poorly understood. Here we show that LINGO-1 is an important negative regulator of this critical process. LINGO-1 is expressed in oligodendrocytes. Attenuation of its function by dominant-negative LINGO-1, LINGO-1 RNA-mediated interference (RNAi) or soluble human LINGO-1 (LINGO-1-Fc) leads to differentiation and increased myelination competence. Attenuation of LINGO-1 results in downregulation of RhoA activity, which has been implicated in oligodendrocyte differentiation. Conversely, overexpression of LINGO-1 leads to activation of RhoA and inhibition of oligodendrocyte differentiation and myelination. Treatment of oligodendrocyte and neuron cocultures with LINGO-1-Fc resulted in highly developed myelinated axons that have internodes and well-defined nodes of Ranvier. The contribution of LINGO-1 to myelination was verified in vivo through the analysis of LINGO-1 knockout mice. The ability to recapitulate CNS myelination in vitro using LINGO-1 antagonists and the in vivo effects seen in the LINGO-1 knockout indicate that LINGO-1 signaling may be critical for CNS myelination.

BAFF (B cell-activating factor belonging to the TNF family) is a cell survival and maturation factor for B cells, and overproduction of BAFF is associated with systemic autoimmune disease. BAFF binds to three receptors, BAFF-R, transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), and B cell maturation Ag (BCMA). Using specific mAbs, BAFF-R was found to be the predominant BAFF receptor expressed on peripheral B cells, in both humans and mice, and antagonist mAbs to BAFF-R blocked BAFF-mediated costimulation of anti-μ responses. The other BAFF receptors showed a much more restricted expression pattern, suggestive of specialized roles. BCMA was expressed by germinal center B cells, while TACI was expressed predominantly by splenic transitional type 2 and marginal zone B cells, as well as activated B cells, but was notably absent from germinal center B cells. BAFF was also an effective costimulator for T cells, and this costimulation occurs entirely through BAFF-R. BAFF-R, but not TACI or BCMA, was expressed on activated/memory subsets of T cells, and T cells from BAFF-R mutant A/WySnJ mice failed to respond to BAFF costimulation. Thus, BAFF-R is important not only for splenic B cell maturation, but is the major mediator of BAFF-dependent costimulatory responses in peripheral B and T cells.