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At least 451 records · Page 25Linked to original sources
PubMed1998

Inducible gene targeting in mice using the Cre/lox system.

Molecular techniques now allow the design of precise genetic modifications in the mouse. Not only can defined nucleotide changes be engineered into the genome of the mouse, but genetic switches can be designed to target expression or ablation of any gene (for which basic molecular information is available) to any tissue at any defined time. These strategies promise to contribute substantially to an increased understanding of individual gene function in development and pathogenesis. A powerful tool, both for the design of such genetic switches and for speeding the creation of gene-modified animals, is the Cre site-specific DNA recombinase of bacteriophage P1. Precise DNA rearrangements and genetic switches can be efficiently generated in a straightforward manner using Cre recombinase. In conjunction with inducible systems for controlling Cre expression and function, these recombination-based strategies are likely to have a profound impact on developmental biology and the generation of useful animal models of human disease.

Animals↗
PubMed1991

Tissue-specific and developmental regulation of rod opsin chimeric genes in transgenic mice.

Chimeric gene fusions between 4.4 kb of rod opsin 5' flanking sequence fused to a diphtheria toxin gene and 4.4 kb or 500 bp of rod opsin 5' flanking sequence fused to the E. coli IacZ gene were used to generate transgenic mice for analysis of cell type-specific expression and temporal and spatial distribution of reporter gene product during retinal development. Opsin-diphtheria toxin transgene expression evoked photoreceptor-specific cell death. The 4.4 kb opsin-IacZ transgene followed temporal and spatial gradients of expression that approximate opsin expression. The 500 bp opsin fragment targeted expression to photoreceptors, but expression was weaker and nonuniform, suggesting that elements located upstream may be required for enhanced and uniform spatial expression.

Animals↗
PubMed2001

Use of red fluorescent protein from Discosoma sp. (dsRED) as a reporter for plant gene expression.

The suitability of the recently described red fluorescent protein dsRED from reef corals for use as a reporter in plant molecular biology was investigated. Based on the clone pDSRED (Clontech), plant expression vectors were constructed for constitutive dsRED expression in the cytosol, the endoplasmic reticulum and the vacuole. Fluorescence microscopy of tobacco BY2 suspension culture cells transiently expressing the plant vectors generated proved that cytosolic expression of the dsRED gives rise to readily detectable levels of red fluorescence, whereas expression in the ER was poor. Vacuolar dsRED expression did not result in any significant fluorescence. dsRED transgenic tobacco SR1 plants were generated to test the sensitivity of dsRED as a reporter in an autofluorescent background, and to identify the possible impact of the introduced fluorescent protein on morphogenesis, plant development and fertility. During the transformation and regeneration phase plants did not show any abnormalities, indicating that dsRED is not interfering with plant development and morphogenesis. Regenerated plants were analysed by PCR, Western blot and fluorescence microscopy for the presence and expression of the transferred genes. The filter sets chosen for fluorescence microscopy proved to be able to block the red chlorophyll fluorescence completely, allowing specific dsRED detection. Best expression levels were obtained with dsRED targeted to the cytosol or chloroplasts. ER-targeted expression of dsRED also gave rise to readily detectable fluorescence levels, whereas vacuolar expression yielded no fluorescence. dsRED transgenic plant lines expressing the protein in the cytosol, ER or chloroplast proved to be fertile. Seed set and germination were normal, except that the seeds and seedlings maintained the red fluorescence phenotype.

Animals↗
PubMed1998

Targeting the receptor-Gq interface to inhibit in vivo pressure overload myocardial hypertrophy.

Hormones and neurotransmitters may mediate common responses through receptors that couple to the same class of heterotrimeric guanine nucleotide-binding (G) protein. For example, several receptors that couple to Gq class proteins can induce cardiomyocyte hypertrophy. Class-specific inhibition of Gq-mediated signaling was produced in the hearts of transgenic mice by targeted expression of a carboxyl-terminal peptide of the alpha subunit Galphaq. When pressure overload was surgically induced, the transgenic mice developed significantly less ventricular hypertrophy than control animals. The data demonstrate the role of myocardial Gq in the initiation of myocardial hypertrophy and indicate a possible strategy for preventing pathophysiological signaling by simultaneously blocking multiple receptors coupled to Gq.

Angiotensin II↗
PubMed2002

[Cloning, expression and biological activity identification of a cDNA encoding the extracellular region of human b7-2].

As one important member of B7/CD28/CTLA-4 costimulatory signal pathway, B7-2 molecule plays a critical role in regulating T-cell response. In order to further explore its effects on regulation of T cell activation, proliferation and associated signal pathways, the cDNA encoding extracellular region of human B7-2 was amplified via PCR and subcloned into some prokaryotic expression vectors to express target protein in host strains. The expressed protein was identified with Western blot and MTT. Results showed that after screening, the expression level of the protein of interest attained the yield of over 20% total bacterial protein by using pGEX-4T-2 vector and E. coli BL21 (DE3)-CodonPlus-RIL host cells. The recombinant protein could specially react with B7-2 McAb and could stimulate T-cell proliferation combined with anti-CD3 antibody. In conclusion, the recombinant protein was bioactive, therefore the study will make it possible for the research of relationship between B7-2 structure and its function.

Antigens, CD↗
PubMed2003

[Inhibition of HBV DNA replication and expression in 2.2.15 hepatoma cells infected with AFP-mediated HBX antisense RNA].

OBJECTIVE: To study the specific expression of the antisense RNA against hepatitis B virus X (HBX) gene in hepatoblastoma cell line and its anti -HBV activity. METHODS: HBX gene (nt.1370-1827) was amplified by PCR, then cloned into EB virus vector pEBAF which contained human alpha-fetoprotein promoter and enhancer. After transfected into 2.2.15 hepatoma cells and ECV304 human endothelial cells by lipofectin, northern blot, ELISA and real-time qualitative PCR were carried out to assay the expression of HBX mRNA, HBV antigens and HBV DNA level, respectively. RESULTS: The HBX antisense RNA expression vector pEBAF-as-HBX which could be expressed specifically in 2.2.15 hepatoblastoma cells was successfully constructed. Both HBV DNA level and the expressions of hepatitis B virus surface antigen (HBsAg) and e antigen (HBeAg) in 2.2.15 hepatoblastoma cells were inhibited by pEBAF-as-HBX. Compared with those in sense control (pEBAF-s-HBX), the inhibitory rates of HBsAg, HBeAg, and HBV DNA were 37.9%, 36.8%, and 25%, respectively. CONCLUSIONS: The pEBAF-as-HBX expression vector may lead to targeted-expression of HBX antisense RNA in hepatoma cells and shows great inhibition effect on HBV.

Animals↗
PubMed1998

Functional dissociation between local and systemic immune response during anti-melanoma peptide vaccination.

Peptide vaccination against tumor Ags can induce powerful systemic CTL responses. However, in the majority of patients, no tumor regression is noted. To study this discrepancy, we analyzed CTL reactivity in a melanoma patient (F001) vaccinated with g209-2M peptide, a single residue variant of gp100(209-217). G209/g209-2M-reactive CTL were identified in post- but not prevaccination PBL. Limiting dilution analysis identified one predominant CTL clone (C1-35), with TCR Vbeta6s2, recognizing g209/HLA-A*0201-expressing targets. Additionally, two autologous melanoma lines (F001TU-3 and -4) and 20 separate tumor-infiltrating lymphocyte cultures were generated from a fine needle aspirate of a metastatic lesion progressing after initial response to vaccination. Both F001TU did not express gp100 and were not recognized by C1-35. Loss of gp100 by F001TU correlated with a marked reduction of gp100 expression in the same metastatic lesion compared with prevaccination. Thus, ineffectiveness of C1-35 and tumor progression could be best explained by loss of target Ag expression. Interestingly, 12 of 20 tumor-infiltrating lymphocyte cultures recognized F001TU, but none demonstrated g209/g209-2M reactivity, suggesting a functional dissociation between systemic and local immune response. This study suggests that vaccination effects must be analyzed in the target tissue, rather than in the systemic circulation alone.

Amino Acid Sequence↗
PubMed2002

Borna disease virus accelerates inflammation and disease associated with transgenic expression of interleukin-12 in the central nervous system.

Targeted expression of biologically active interleukin-12 (IL-12) in astrocytes of the central nervous system (CNS) results in spontaneous neuroimmunological disease of aged mice. Borna disease virus (BDV) can readily multiply in the mouse CNS but does not trigger disease in most strains. Here we show that a large percentage of IL-12 transgenic mice developed severe ataxia within 5 to 10 weeks after infection with BDV. By contrast, no disease developed in mock-infected IL-12 transgenic and wild-type mice until 4 months of age. Neurological symptoms were rare in infected wild-type animals, and if they occurred, these were milder and appeared later. Histological analyses showed that the cerebellum of infected IL-12 transgenic mice, which is the brain region with strongest transgene expression, contained large numbers of CD4(+) and CD8(+) T cells as well as lower numbers of B cells, whereas other parts of the CNS showed only mild infiltration by lymphocytes. The cerebellum of diseased mice further showed severe astrogliosis, calcifications and signs of neurodegeneration. BDV antigen and nucleic acids were present in lower amounts in the inflamed cerebellum of infected transgenic mice than in the noninflamed cerebellum of infected wild-type littermates, suggesting that IL-12 or IL-12-induced cytokines exhibited antiviral activity. We propose that BDV infection accelerates the frequency by which immune cells such as lymphocytes and NK cells enter the CNS and then respond to IL-12 present in the local milieu causing disease. Our results illustrate that infection of the CNS with a virus that is benign in certain hosts can be harmful in such normally disease-resistant hosts if the tissue is unfavorably preconditioned by proinflammatory cytokines.

Animals↗
PubMed1998

[Retroviral-mediated transcriptional regulatory elements of alpha-fetoprotein gene directed specific expression of interleukin-2 in murine hepatoma].

OBJECTIVE: To target expression of interleukin-2 in alpha-fetoprotein (AFP) positive hepatocellular carcinoma. METHODS: A retroviral vector pDOR-mAFP-IL2 was constructed by coupling the transcriptional regulatory elements (TREs) of murine AFP gene with interleukin-2 cDNA in a retroviral vector. RESULTS: Subsequent to retroviral infection, expression of IL-2 could be detected in an AFP positive murine hepatoma cell line Hepa1-6 but not in psi 2, an AFP negative murine fibroblast cell line. CONCLUSIONS: Our results indicate that specific expression of IL-2 in hepatoma cell can be achieved by using of AFP TREs to control the expression of IL-2, and the retroviral vector which we have constructed may be used for further experimental studies on in vivo gene therapy.

Animals↗
PubMed1983

Cell surface expression of cytotoxic T lymphocyte-defined, AKR/Gross leukemia virus-associated tumor antigens by normal AKR.H-2b splenic B cells.

We previously described a system in which H-2Kb-restricted C57BL/6 (B6) cytotoxic T lymphocytes (CTL) could be raised that were specific for tumors, such as the thymic lymphoma AKR.H-2b SL1, that were induced by endogenous AKR/Gross murine leukemia virus and that expressed the Gross cell surface antigen. In this study, certain normal lymphoid cells from AKR.H-2b mice were also found to express target antigens defined by such anti-AKR/Gross virus CTL. AKR.H-2b spleen, but surprisingly not thymus, cells stimulated the production of anti-AKR/Gross virus CTL when employed at either the in vivo priming phase or the in vitro restimulation phase of anti-viral CTL induction. This selective stimulation by spleen vs thymus cells was not dependent on the age of the mice over the range (3 to 28 wk) tested. Both AKR.H-2b spleen and thymus cells, however, were able to stimulate the generation of H-2-restricted B6 anti-AKR minor histocompatibility (H) antigen-specific CTL. Thus, AKR.H-2b spleen cells appeared to display the same sets (minor H and virus-associated) of cell surface antigens recognized by CTL as the AKR.H-2b SL1 tumor, whereas AKR.H-2b thymocytes were selectively missing the virus-associated target antigens, a situation analogous to that of cl. 18-5, a variant subclone of AKR.H-2b SL1 insusceptible to anti-AKR/Gross virus CTL. Like AKR.H-2b thymocytes, neither AKR spleen cells or thymocytes nor B6.GIX + thymocytes were able to stimulate the generation of anti-AKR/Gross virus CTL from primed B6 responder cell populations. In contrast, both T cell-enriched and B cell-enriched preparations derived from AKR.H-2b spleen cells were able to stimulate at the in vitro phase of induction, although B cell-enriched preparations were considerably more efficient. The discordant results obtained with AKR.H-2b spleen cells vs thymocytes were confirmed and extended in experiments in which these cells were employed as target cells to directly assess the cell surface expression of virus-associated, CTL-defined antigens. Thus, AKR.H-2b spleen cells, but not thymocytes, were recognized by anti-AKR/Gross virus CTL when fresh normal cells were tested as unlabeled competitive inhibitors, or when mitogen blasts were tested as labeled targets. Fresh or lipopolysaccharide-stimulated B cell-enriched spleen cells were as efficiently recognized as unseparated spleen cell preparations. Unexpectedly, fresh or Lens culinaris hemagglutinin-stimulated T cell-enriched spleen cell preparations, although susceptible to anti-minor H CTL, were almost as poor as targets for anti-viral CTL as were thymocytes. Together, these results demonstrate the H-2-restricted expression of CTL-defined, endogenous, AKR/Gross virus-associated target antigens by normal AKR.H-2b splenic B cells.(ABSTRACT TRUNCATED AT 400 WORDS)

AKR murine leukemia virus↗
PubMed2001

Biliverdin reductase-induced phytochrome chromophore deficiency in transgenic tobacco.

Targeted expression of mammalian biliverdin IXalpha reductase (BVR), an enzyme that metabolically inactivates linear tetrapyrrole precursors of the phytochrome chromophore, was used to examine the physiological functions of phytochromes in the qualitative short-day tobacco (Nicotiana tabacum cv Maryland Mammoth) plant. Comparative phenotypic and photobiological analyses of plastid- and cytosol-targeted BVR lines showed that multiple phytochrome-regulated processes, such as hypocotyl and internode elongation, anthocyanin synthesis, and photoperiodic regulation of flowering, were altered in all lines examined. The phytochrome-mediated processes of carotenoid and chlorophyll accumulation were strongly impaired in plastid-targeted lines, but were relatively unaffected in cytosol-targeted lines. Under certain growth conditions, plastid-targeted BVR expression was found to nearly abolish the qualitative inhibition of flowering by long-day photoperiods. The distinct phenotypes of the plastid-targeted BVR lines implicate a regulatory role for bilins in plastid development or, alternatively, reflect the consequence of altered tetrapyrrole metabolism in plastids due to bilin depletion.

Agrobacterium tumefaciens↗
PubMed2001

The roles of activins in repair processes of the skin and the brain.

A recent study from our laboratory demonstrated a strong upregulation of activin expression during cutaneous wound healing. To further analyze the role of activin A in skin morphogenesis and wound repair, we generated transgenic mice that overexpress activin A under the control of the keratin 14 promoter. The latter targets expression of transgenes to the basal, proliferating layer of the epidermis. Hetero- as well as homozygous transgenic animals were viable and fertile. However, they were smaller than non-transgenic littermates and they had smaller ears and shorter tails. Histological analysis of their skin revealed dermal hyperthickening, mainly due to the replacement of fatty tissue by connective tissue, and an increase in suprabasal, partially differentiated epidermal layers. After cutaneous injury, a strong enhancement of granulation tissue formation was observed. Furthermore, the extent of re-epithelialization was increased in some of the wounds. These data demonstrate that activin A is a potent stimulator of the wound healing process. Using an in vivo model of local brain injury, we found that activin A also plays a significant role in the early cellular response to neuronal damage. Expression of activin mRNA and protein is markedly upregulated within a few hours of injury. If applied exogenously, recombinant activin A is capable of rescuing neurons from acute cell death. Studying the interaction between bFGF, a well-established neuroprotective agent, which is currently being tested in stroke patients, and activin A, we arrived at the unexpected conclusion that it is the strong induction of activin A by bFGF which endows the latter with its beneficial actions in patients. These findings suggest that the development of substances directly targeting activin expression or receptor binding should offer new possibilities in the acute treatment of stroke and brain trauma.

Activins↗
PubMed1997

Identification of epidermal growth factor receptor and c-erbB2 pathway inhibitors by correlation with gene expression patterns.

BACKGROUND: Growth factor receptor-signaling pathways are potentially important targets for anticancer therapy. The interaction of anticancer agents with specific molecular targets can be identified by correlating target expression patterns with cytotoxicity patterns. We sought to identify new agents that target and inhibit the activity of the epidermal growth factor (EGF) receptor and of c-erbB2 (also called HER2 or neu), by correlating EGF receptor, transforming growth factor (TGF)-alpha (a ligand for EGF receptor), and c-erbB2 messenger RNA (mRNA) expression levels with the results of cytotoxicity assays of the 49000 compounds in the National Cancer Institute (NCI) drug screen database. METHODS: The levels of mRNAs were measured and used to generate a molecular target database for the 60 cell lines of the NCI anticancer drug screen. The computer analysis program, COMPARE, was used to search for cytotoxicity patterns in the NCI drug screen database that were highly correlated with EGF receptor, TGF-alpha, or c-erbB2 mRNA expression patterns. The putative EGF receptor-inhibiting compounds were tested for effects on basal tyrosine phosphorylation, in vitro EGF receptor tyrosine kinase activity, and EGF-dependent growth. Putative ErbB2-inhibiting compounds were tested for effects on antibody-induced ErbB2 tyrosine kinase activity. RESULTS: EGF receptor mRNA and TGF-alpha mRNA levels were highest in cell lines derived from renal cancers, and c-erbB2 mRNA levels were highest in cells derived from breast, ovarian, and colon cancers. Twenty-five compounds with high correlation coefficients (for cytotoxicity and levels of the measured mRNAs) were tested as inhibitors of the EGF receptor or c-erbB2 signaling pathways; 14 compounds were identified as inhibitors of these pathways. The most potent compound, B4, inhibited autophosphorylation (which occurs following activation) of ErbB2 by 50% in whole cells at 7.7 microM. CONCLUSIONS: Novel EGF receptor or c-erbB2 pathway inhibitors can be identified in the NCI drug screen by correlation of cytotoxicity patterns with EGF receptor or c-erbB2 mRNA expression levels.

Antineoplastic Agents↗
PubMed1998

Discrete stages of anterior pituitary differentiation recapitulated in immortalized cell lines.

Targeted expression of oncogenes in transgenic mice can immortalize specific cell types to serve as valuable cultured model systems. Utilizing promoter regions from pituitary genes activated in the gonadotrope/thyrotrope lineage at discrete stages of development, we have demonstrated that targeted oncogene expression in transgenic mice can produce cell lines representing sequential stages of differentiation. Each cell line expresses a specific subset of the genes denoting differentiated function such as the subunits of the glycoprotein hormones, hormone receptors, and transcriptional regulatory proteins. These model systems have allowed detailed investigations into molecular and cellular mechanisms otherwise inaccessible in vivo or in complex primary pituitary cell cultures.

Animals↗
PubMed1978

Sensitization of lymphocytes against pooled allogeneic cells. I. Generation of cytotoxicity against autologous human lymphoblastoid cell lines.

Lymphocytes sensitized in vitro to a pool of X-irradiated allogeneic normal lymphocytes from 20 individuals develop cytotoxic activity for autologous human lymphoblastoid cells (LCL). Whereas pool sensitized T lymphocytes lyse autologous LCL cells, they fail to lyse autologous B-enriched or T-enriched normal target cells nor autologous phytohemagglutinin (PHA) blasts. In contrast to pool sensitization, stimulation with normal cells of single allogeneic individuals rarely led to development of cytotoxicity against autologous LCL cells. We conclude that human Epstein-Barr virus transformed LCL cells express target antigens cross-reactive with allogeneic target antigens expressed on normal cells and that sensitization with a pool of allogeneic cells is an effective means of generating effector cells directed against autologous abnormal cells.

B-Lymphocytes↗
PubMed1990

Assessment of the specificity of cytotoxic T lymphocytes for the nucleoprotein of Pichinde virus using recombinant vaccinia viruses.

Pichinde virus (PV) infection of mice results in induction of a strong H-2 restricted, virus-specific cytotoxic T lymphocyte (CTL) response and rapid clearance of the virus. To define the specificities of CTL induced by PV infection, we constructed vaccinia virus recombinants containing cloned cDNAs corresponding to full-length (VVNP) and a truncated form (VVNP 51-561) of the nucleoprotein (NP) gene of PV. Radioimmunoprecipitation analysis of infected cell lysates indicated that VVNP expressed a PV-specific product identical in size to that of authentic NP, while vaccinia virus recombinants containing truncated NP produced a polypeptide consistent with the synthesis of amino acids 51-561 of Pichinde virus NP. Interestingly, cells infected with VVNP synthesized easily detectable, but much lower levels of nucleoprotein relative to both PV and VVNP51-561. Primary virus-specific CTL induced in three different strains of inbred mice following intravenous infection with PV were able to lyse syngeneic target cells infected with PV but did not markedly lyse syngeneic targets expressing full-length or truncated NP following recombinant vaccinia virus infection. Similarly, secondary anti-PV specific CTL generated following in vitro restimulation by PV or selectively restimulated with vaccinia recombinants did not significantly lyse target cells expressing NP. Further, infection of mice with VVNP and VVNP51-561 did not induce CTLs specific for PV and did not prime mice for the generation of memory anti-PV CTL in vivo. These results suggest that PV gene products other than NP, such as the GPC or L protein, contain the major target epitope(s) recognized by PV-specific CTL.

Animals↗
PubMed2026

A single-cell atlas of multiple myeloma defines malignant archetypes and proliferative states.

Multiple myeloma (MM) is a plasma-cell malignancy with extensive genomic and transcriptional heterogeneity, limiting disease classification and precision therapy. Here we generated a clinically annotated, population-scale, single-cell atlas of MM from 341 individuals spanning the disease and treatment continuum. We identified five recurrent malignant transcriptional archetypes and an orthogonal proliferative program associated with genomic features, therapeutic resistance and clinical outcomes. Validation in the independent CoMMpass cohort demonstrated robustness, prognostic relevance and portability across platforms. We developed a single-cell, target-discovery pipeline prioritizing malignant enrichment, cell-type specificity and tissue restriction, identifying FCRL2 as a plasma-restricted or B cell-lineage-restricted surface target expressed by malignant plasma cells. FCRL2-targeted chimeric antigen receptor T cells demonstrated antigen-specific activity in vitro and survival benefit in vivo. Together, these data provide a clinically actionable blueprint for patient stratification and precision target nomination in plasma-cell malignancies.

Multiple Myeloma↗
PubMed1988

Increased resistance to non-MHC-restricted cytotoxicity related to HLA A, B expression. Direct demonstration using beta 2-microglobulin-transfected Daudi cells.

Experiments in several laboratories have shown that target susceptibility to NK and lymphokine-activated killer (LAK) cytotoxicity is inversely correlated with the target expression of HLA Class I molecules. We present the first direct evidence, obtained by gene transfection, that target cell HLA, A, B expression increases the resistance to the "so-called" non-MHC-restricted cytotoxicity. We have co-transfected, by electroporation, the human beta 2-microglobulin gene and the gene carrying the resistance to geneticin into Daudi cell line. Geneticin selection in culture followed by FACS sorting on the basis of strong positivity with the mAb W6/32 (which is specific for the HLA class I H chain associated to beta 2-microglobulin) have led to the establishment of a HLA+ Daudi cell line permanently expressing HLA A10, A11, and B17 molecules. Studies were performed in vitro to evaluate the susceptibility of these cells to either NK and LAK cytotoxicity. The HLA class I+ Daudi cells exhibit an increased resistance to killing by non-MHC-restricted killer cells (both NK and LAK) as compared with their HLA-Daudi counterpart.

Cytotoxicity, Immunologic↗