Showing papers by "David Baltimore published in 2002"

Germline Transmission and Tissue-Specific Expression of Transgenes Delivered by Lentiviral Vectors

Abstract: 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.

The IκB-NF-κB Signaling Module: Temporal Control and Selective Gene Activation

Abstract: Nuclear localization of the transcriptional activator NF-κB (nuclear factor κB) is controlled in mammalian cells by three isoforms of NF-κB inhibitor protein: IκBα, -β, and -ɛ Based on simplifying reductions of the IκB–NF-κB signaling module in knockout cell lines, we present a computational model that describes the temporal control of NF-κB activation by the coordinated degradation and synthesis of IκB proteins The model demonstrates that IκBα is responsible for strong negative feedback that allows for a fast turn-off of the NF-κB response, whereas IκBβ and -ɛ function to reduce the system's oscillatory potential and stabilize NF-κB 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

CARD11 mediates factor-specific activation of NF-κB by the T cell receptor complex

Abstract: NF‐κB is a critical target of signaling downstream of the T cell receptor (TCR) complex, but how TCR signaling activates NF‐κB is poorly understood. We have developed an expression cloning strategy that can identify catalytic and noncatalytic molecules that participate in different pathways of NF‐κB activation. Screening of a mouse thymus cDNA library yielded CARD11, a membrane‐associated guanylate kinase (MAGUK) family member containing CARD, PDZ, SH3 and GUK domains. Using a CARD‐deleted variant of CARD11 and RNA interference (RNAi), we demonstrate that CARD11 mediates NF‐κB activation by αCD3/αCD28 cross‐linking and PMA/ionomycin treatment, but not by TNFα or dsRNA. CARD11 is not required for TCR‐mediated induction of NFAT or AP‐1. CARD11 functions upstream of the IκB‐kinase (IKK) complex and cooperates with Bcl10 in a CARD domain‐dependent manner. RNAi‐rescue experiments suggest that the CARD, coiled‐coil, SH3 and GUK domains of CARD11 are critical for its signaling function. These results implicate CARD11 in factor‐ specific activation of NF‐κB by the TCR complex and establish a role for a MAGUK family member in antigen receptor signaling.

Cooperation of multiple signaling pathways in CD40-regulated gene expression in B lymphocytes

Abstract: CD40/CD40L interaction is essential for multiple biological events in T dependent humoral immune responses, including B cell survival and proliferation, germinal center and memory B cell formation, and antibody isotype switching and affinity maturation. By using high-density microarrays, we examined gene expression in primary mouse B lymphocytes after multiple time points of CD40L stimulation. In addition to genes involved in cell survival and growth, which are also induced by other mitogens such as lipopolysaccharide, CD40L specifically activated genes involved in germinal center formation and T cell costimulatory molecules that facilitate T dependent humoral immunity. Next, by examining the roles of individual CD40-activated signal transduction pathways, we dissected the overall CD40-mediated response into genes independently regulated by the individual pathways or collectively by all pathways. We also found that gene down-regulation is a significant part of the overall response and that the p38 pathway plays an important role in this process, whereas the NF-κB pathway is important for the up-regulation of primary response genes. Our finding of overlapping independent control of gene expression modules by different pathways suggests, in principle, that distinct biological behaviors that depend on distinct gene expression subsets can be manipulated by targeting specific signaling pathways.

Essential roles of the κ light chain intronic enhancer and 3′ enhancer in κ rearrangement and demethylation

Abstract: The kappa intronic (MiE(kappa)) and 3' (3'E(kappa)) enhancers are both quantitatively important to, but not essential for, immunoglobulin kappa rearrangement. To determine the functional redundancy between these two enhancers, B cells derived from mutant embryonic stem cells--in which both MiE(kappa) and 3'E(kappa) were deleted on both kappa alleles--were analyzed for kappa rearrangement. Our findings indicate that these double-mutant B cells have essentially no kappa rearrangement but do rearrange and express lambda. Therefore, these two kappa enhancers share essential roles in activating V(kappa)J(kappa) rearrangement. Our findings also indicate that the two kappa enhancers play overlapping and distinct roles in the demethylation of kappa in B cells.

The combined absence of NF-κB1 and c-Rel reveals that overlapping roles for these transcription factors in the B cell lineage are restricted to the activation and function of mature cells

Abstract: Transcription factors NF-KB1 and c-Rel, individually dispensable during embryogenesis, serve similar, yet distinct, roles in the function of mature hemopoietic cells. Redundancy among Rel/ NF-KB family members prompted an examination of the combined roles of c-Rel and NF-KB1 by using mice that lack both proteins. Embryonic development and the maturation of hemopoietic progenitors were unaffected in nfkb1(-/-)c-rel(-/-) mice. Peripheral T cell populations developed normally, but follicular, marginal zone, and CD5(+) peritoneal B cell populations all were reduced. In culture, a failure of mitogen-stimulated nfkb1(-/-)c-rel(-/-) B cells to proliferate was caused by a cell cycle defect in early G(1) that prevented growth. In vivo, defects in humoral immunity and splenic architecture seen in nfkbl(-/-) and c-rel(-/-) mice were exacerbated in the double mutant mice. These findings demonstrate that in the B lineage overlapping roles for NF-K81 and c-Rel appear to be restricted to regulating the activation and function of mature cells.

Method for the generation of antigen-specific lymphocytes

Abstract: The invention provides systems and methods for the generation of lymphocytes having a unique antigen specificity. In a preferred embodiment, the invention provides methods of virally infecting cells from bone marrow with one or more viral vectors that encode antigen-specific T cell receptors. The resulting lymphocytes, and in particular, T cells express the T cell receptor (TCR) that was introduced. The lymphocytes generated can be used for a variety of therapeutic purposes including the treatment of various cancers and the generation of a desired immune response to viruses and other pathogens. The resulting cells develop normally and respond to antigen both in vitro and in vivo. It is possible to modify the function of lymphocytes by using stem cells from different genetic backgrounds. Thus the system constitutes a powerful tool to generate desired lymphocyte populations both for research and therapy. Applications of this technology include treatments for infectious diseases, such as HIV/AIDS, cancer therapy, allergy, and autoimmune disease.

Requirement for the NF-κB Family Member RelA in the Development of Secondary Lymphoid Organs

Abstract: The transcription factor nuclear factor (NF)-κB has been suggested to be a key mediator of the development of lymph nodes and Peyer's patches. However, targeted deletion of NF-κB/ Rel family members has not yet corroborated such a function. Here we report that when mice lacking the RelA subunit of NF-κB are brought to term by breeding onto a tumor necrosis factor receptor (TNFR)1-deficient background, the mice that are born lack lymph nodes, Peyer's patches, and an organized splenic microarchitecture, and have a profound defect in T cell–dependent antigen responses. Analyses of TNFR1/RelA-deficient embryonic tissues and of radiation chimeras suggest that the dependence on RelA is manifest not in hematopoietic cells but rather in radioresistant stromal cells needed for the development of secondary lymphoid organs.

Generation of functional antigen-specific T cells in defined genetic backgrounds by retrovirus-mediated expression of TCR cDNAs in hematopoietic precursor cells

Abstract: We have developed an alternative to transgenesis for producing antigen-specific T cells in vivo. In this system, clonal naive T cells with defined antigen specificity are generated by retrovirus-mediated expression of T cell antigen receptor cDNAs in RAG1-deficient murine hematopoietic precursor cells. These T cells can be stimulated to proliferate and produce cytokines by exposure to antigen in vitro, and they become activated and expand in vivo after immunization. IL-2-deficient T cells generated by this technique show decreased proliferation and cytokine production, both of which can be rescued by exogenous addition of this growth factor. Thus, retrovirus-mediated expression of T cell antigen receptor cDNAs in hematopoietic precursor cells permits the rapid and efficient analysis of the life history of antigen-specific T cells in different genetic backgrounds and may allow for the long-term production of antigen-specific T cells with different functional properties for prophylactic and therapeutic purposes.

Method for expression of small RNA molecules within a cell

Abstract: The invention provides methods and compositions for the expression of small RNA molecules within a cell using a lentiviral vector. The methods can be used to express doubles stranded RNA complexes. Small interfering RNA (siRNA) can be expressed using the methods of the invention within a cell, which are capable of down regulating the expression of a target gene through RNA interference. A variety of cells can be treated according to the methods of the invention including embryos, embryogenic stem cells, allowing for the generation of transgenic animals or animals constituted partly by the transduced cells that have a specific gene or a group of genes down regulated.

Inhibition of DNA Binding by NF-κB with Pyrrole-Imidazole Polyamides

Abstract: Synthetic ligands that bind to predetermined DNA sequences will offer a chemical approach to gene regulation if inhibition of a broad range of transcription factors can be achieved. NF-κB is a transcription factor that regulates a multitude of genes, including those involved in immune, inflammatory, and anti-apoptotic responses. NF-κB binds as heterodimer predominantly in the major groove. We report the design of polyamides that bind in the minor groove and target overlapping portions of an NF-κB binding site (5‘-GGGACTTTCC-3‘). We find that compounds that target the 5‘-GGGACT-3‘ portion of the site can inhibit DNA binding by NF-κB while those that target the 5‘-ACTTTCC-3‘ portion do not. Addition of NF-κB to the list of protein−DNA complexes that can be disrupted by minor groove binding ligands potentially increases the utility of polyamides as regulators of gene expression.

Steering a course to an AIDS vaccine.

Abstract: F or more than 20 years, AIDS has been progressing relentlessly and predictably while medical technology has been stymied in its effort to provide a fix. We do have effective drugs, but they treat the infection at great expense and with great difficulty. And we know what will do the job: a safe and effective vaccine. After all, vaccines stopped polio and hepatitis B. The difference is that those viruses are highly sensitive to antibody killing, so the vaccines needed only to induce antibodies. But HIV, the unquestionable cause of AIDS, has evolved to elude antibody killing, thwarting our attempts to induce a broadly protective antibody response, even in animals. A test of an antibody-based vaccine is being run by an optimistic company, but few experts give it much chance of success. Are we powerless? No. A century of study of immunology and protein structure gives us hope that there are ways of designing immunogens that will work. So we examine each detail of the virus's structure, trying to find chinks in its armor where an antibody might penetrate. But the immunologists hold out a different hope: that there is a second type of immunity—the activity of killer T cells—that can clear some viral infections and help antibodies clear others. Maybe we could devise a way to use this arm of our immune systems against HIV. Over 10 years of research has been devoted to this hope, and great progress has been made. But we are still at an early stage; testing of the most extensively evaluated candidate vaccine was recently halted because it is not giving sufficient evidence of immunogenicity. ![Figure][1] 20 Years of HIV/AIDSSOURCE: UNAIDS; ILLUSTRATION: C. FABER SMITH At present, we have a pipeline of potential stimulants of T cell-based immunity feeding into early-stage testing. However, little has been evaluated in humans even for basic safety (Phase I trials). The most promising T cell stimulant is a DNA vaccine followed by a viral vector; it is still in an early phase of testing. Thus, should all go well, we might have a vaccine in 5 years, but things rarely go so well in this difficult business. Few will be surprised if it takes 10 years to get to a licenced vaccine. One big fear now is that we will be able to stimulate T cell immunity, but the virus will quickly elude it by changing its structure a bit. Already in monkeys and humans there is evidence of viral escape from T cell immunity. For reasons that still elude immunologists, even if you stimulate immunity with a complex immunogen, the T cells focus their response on just one simple peptide structure, making it easy for the virus to mutate to resistance. So the T cell route to immunity may yet be a very bumpy road. Are we appropriately organized to respond to this devastating epidemic? A plus is that the U.S. government is putting more money into AIDS research, and specifically into vaccine development, than the rest of the world combined. But the academic community, while taking the money, is still working on a business-as-usual basis. By contrast, the National Institutes of Health (NIH) itself has set up an integrated unit dedicated to making an HIV vaccine. It combines protein structure determination, immunology, vaccine candidate development, primate testing, and clinical assessment. Leadership in the vaccine effort at the government level has been diffuse and invisible recently. When Harold Varmus was NIH director, the effort had high-level patronage and constant visibility. There is now new NIH leadership, and we can only hope that the new director will understand that he can have no higher priority than to deal with the AIDS epidemic. It threatens world stability, it kills Americans, and it is the greatest threat to those who can least understand the need to take precautions against infection: the poor, the underserved, and the populations of underdeveloped countries. Next week, the biannual International AIDS Meeting will take place. There is unlikely to be any exciting news on the vaccine front, because progress is slow. It is important to realize that vaccine research is intrinsically slow because it takes a long time to know whether a trial has been successful. But we ought to make every effort to provide the leadership and vision to ensure that this inevitably protracted process will proceed at the fastest possible pace. [1]: pending:yes