Activation of mammalian innate and acquired immune responses must be tightly regulated by elaborate mechanisms to control their onset and termination. MicroRNAs have been implicated as negative regulators controlling diverse biological processes at the level of posttranscriptional repression. Expression profiling of 200 microRNAs in human monocytes revealed that several of them (miR-146a͞b, miR-132, and miR-155) are endotoxin-responsive genes. Analysis of miR-146a and miR-146b gene expression unveiled a pattern of induction in response to a variety of microbial components and proinflammatory cytokines. By means of promoter analysis, miR-146a was found to be a NF-B-dependent gene. Importantly, miR-146a͞b were predicted to base-pair with sequences in the 3 UTRs of the TNF receptor-associated factor 6 and IL-1 receptor-associated kinase 1 genes, and we found that these UTRs inhibit expression of a linked reporter gene. These genes encode two key adapter molecules downstream of Toll-like and cytokine receptors. Thus, we propose a role for miR-146 in control of Toll-like receptor and cytokine signaling through a negative feedback regulation loop involving down-regulation of IL-1 receptor-associated kinase 1 and TNF receptor-associated factor 6 protein levels.immunity ͉ microRNA ͉ LPS ͉ Toll-like receptor

do not readily transform cells upon transfection. An exception is v-Rel which, however, can acutely transform *Tularik Inc. only avian cells in culture. The transforming potential Two Corporate Drive of a RelA splice variant called p65⌬ in rodent cells re-South San Francisco, California 94080 CA 94080 mains a matter of debate (Narajanan et al., 1992; Grimm Department of Biology and Baeuerle, 1994). T. Gilmore (Boston University, Bos- † Massachusetts Institute of Technology ton) reviewed the properties of v-Rel that contribute to 77 Massachusetts Ave.

Studies on mice deficient in nuclear factor kappa B (NF-kappaB) subunits have shown that this transcription factor is important for lymphocyte responses to antigens and cytokine-inducible gene expression. In particular, the RelA (p65) subunit is required for induction of tumor necrosis factor-alpha (TNF-alpha)-dependent genes. Treatment of RelA-deficient (RelA-/-) mouse fibroblasts and macrophages with TNF-alpha resulted in a significant reduction in viability, whereas RelA+/+ cells were unaffected. Cytotoxicity to both cell types was mediated by TNF receptor 1. Reintroduction of RelA into RelA-/- fibroblasts resulted in enhanced survival, demonstrating that the presence of RelA is required for protection from TNF-alpha. These results have implications for the treatment of inflammatory and proliferative diseases.

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.

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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.

Single-cell mouse embryos were infected in vitro with recombinant lentiviral vectors to generate transgenic mice carrying the green fluorescent protein (GFP) gene driven by a ubiquitously expressing promoter. Eighty percent of founder mice carried at least one copy of the transgene, and 90% of these expressed GFP at high levels. Progeny inherited the transgene(s) and displayed green fluorescence. Mice generated using lentiviral vectors with muscle-specific and T lymphocyte-specific promoters expressed high levels of GFP only in the appropriate cell types. We have also generated transgenic rats that express GFP at high levels, suggesting that this technique can be used to produce other transgenic animal species.

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