Yeast expression of animal and plant P450s in optimized redox environments.
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Animals↗
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Results for “vector engineering”
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The essential role of hippocampal CA1 NMDA receptor-dependent synaptic plasticity in spatial memory.
We have produced a mouse strain in which the deletion of the NMDAR1 gene is restricted to the CA1 pyramidal cells of the hippocampus by using a new and general method that allows CA1-restricted gene knockout. The mutant mice grow into adulthood without obvious abnormalities. Adult mice lack NMDA receptor-mediated synaptic currents and long-term potentiation in the CA1 synapses and exhibit impaired spatial memory but unimpaired nonspatial learning. Our results strongly suggest that activity-dependent modifications of CA1 synapses, mediated by NMDA receptors, play an essential role in the acquisition of spatial memories.
Animals↗
Human l1 retrotransposition is associated with genetic instability in vivo.
Retrotransposons have shaped eukaryotic genomes for millions of years. To analyze the consequences of human L1 retrotransposition, we developed a genetic system to recover many new L1 insertions in somatic cells. Forty-two de novo integrants were recovered that faithfully mimic many aspects of L1s that accumulated since the primate radiation. Their structures experimentally demonstrate an association between L1 retrotransposition and various forms of genetic instability. Numerous L1 element inversions, extra nucleotide insertions, exon deletions, a chromosomal inversion, and flanking sequence comobilization (called 5' transduction) were identified. In a striking number of integrants, short identical sequences were shared between the donor and the target site's 3' end, suggesting a mechanistic model that helps explain the structure of L1 insertions.
5' Flanking Region↗
DNA in medicine. The tools. Part II.
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Base Sequence↗
Conjugal transfer of plasmid pTF-FC2 from Agrobacterium to plant cells in the absence of T-DNA borders.
The ability of the plasmid pTF-FC2 to transfer genes into plants was investigated. Using this plasmid as the backbone two plasmids were constructed namely pTD1 and pDER-bar. These plasmids contained, as plant selectable markers, the nptII and the bar genes, respectively. The nptII gene was flanked by the right and left borders and the bar gene was not. Transgenic plants were obtained through the co-cultivation of tobacco leaf discs with the Agrobacterium tumefaciens strain LBA4404(pAL4404)(pDER-bar). Molecular and genetic analysis indicated that the bar gene had been stably integrated into the plant genome and had been inherited in a Mendelian fashion. Integration was shown to be polar and unidirectional and in some cases the entire plasmid was found to have integrated into the plant genome. Interestingly, no plants were generated from tobacco leaf discs that were co-cultivated with the strain C58C1(pMP90)(pTD1).
Agrobacterium tumefaciens↗
Autologous, allogeneic tumor cells or genetically engineered cells as cancer vaccine against melanoma.
Melanoma is a prototype of immunogenic tumor to which various types of immunotherapy have been applied extensively over the past decades. Melanoma vaccines are designed for the purpose of immune modulation and subsequent anti-tumor effects in the process of an active specific immunotherapy. Previous attempts of these vaccines include immunization with whole tumor cells/cell lysates admixed with nonspecific adjuvants. While these vaccines generated enhanced anti-tumor immunity in a subset of patients, some of which showing prolonged survival compared to historical controls, no clinical benefit has so far been demonstrated in a properly controlled phase III study. New-generation melanoma vaccines, which are based on genetic modifications of tumor cells to express cytokines, generated long-lasting systemic anti-tumor immunity in animal models. Translation of these preclinical results primarily into melanoma patients with advanced diseases, shows the potential of these vaccines to induce systemic anti-tumor immune responses and in some instances tumor regression with acceptably low toxicity. Higher efficacy of this novel vaccine approach would be expected when used in a postsurgical adjuvant setting when the tumor load is small. Also other novel vaccine approaches such as dendritic cell-based therapy hold promise for the treatment of melanoma. But the clinical value of all these new approaches has to be analysed in prospectively randomized clinical studies.
Antigens, Neoplasm↗
The genetic engineering of microbial solvent production.
Although the production of acetone and butanol by Clostridium strains was a thriving industrial fermentation process, it is no longer competitive with the chemical synthesis of solvents and has been discontinued. However, studies on the molecular biology of Clostridium strains suggest that genetic engineering for improved solvent production is feasible, and could result in the revival of the industrial fermentation process.
1-Butanol↗
Decision making under the Cartagena Protocol on Biosafety.
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Animals↗
Negative-strand RNA viruses: applications to biotechnology.
The establishment of reverse-genetics techniques to manipulate the genome of negative-strand RNA viruses has contributed enormously to a better understanding of the replication mechanisms and pathogenicity of this group of viruses. The generation of recombinant viruses bearing specific mutations in the coding and noncoding regions of their genomic RNAs now allows the functions in the replicative cycle of specific RNA regions and protein domains of these viruses to be studied. In addition, recombinant negative-strand RNA viruses can now be designed to have specific properties that make them attractive biotechnological tools.
Animals↗
Adaptable modification of adenoviral tropism using a bifunctional ligand protein.
In order to target recombinant adenovirus (AdV), we have developed a new strategy using a fusion ligand protein comprising coxsackievirus/adenovirus receptor (CAR), and the antibody Fc-binding domain from protein A in vitro testing with this ligand shows that it blocks viral gene transduction and, when coupled with anti-ICAM-1 IgG, redirects AdV to endothelial cells that are induced to express ICAM-1. Because the protein A Fc-binding domain will bind to any immunoglobulin, the current strategy can be adapted to target a wide variety of tissues or cells as long as an antibody species that recognizes a membrane marker on target tissue or cell is present. This concept may be further expanded to other viruses that employ peptide receptors. These membrane receptors can be fused to the Fc-binding domain to create a variety of bifunctional ligands for targeting recombinant viruses in gene therapy.
Adenoviridae↗
Cellular transplants as sources for therapeutic agents.
Soluble factors normally produced by cells of the human body are of increasing importance as potential therapeutic agents. Although considerable progress has been made in understanding the etiology and pathogenesis of disease, in developing animal models and newer experimental therapeutics, few discoveries have been translated into clinically effective ways of delivering the multiple therapeutic agents obtained from living mammalian cells. This review examines the use of transplanted cells as alternatives to conventional delivery systems to deliver a variety of protein based therapeutic agents. The chapter begins with a set of questions to establish the complexity and challenges of this form of drug delivery. The following section focuses the discussion on our understanding of genetic engineering, tissue engineering, and some areas of developmental biology as they relate to the development of this nascent field. Much of the discussion has a neuro/endocrine emphasis. The chapter ends by listing the basic ingredients needed to push the use of transplanted cells toward medical practice and some general comments about future developments.
Animals↗
Highly divergent amino termini of the homologous human ALR and yeast scERV1 gene products define species specific differences in cellular localization.
The yeast scERV1 gene product is involved in the biogenesis of mitochondria and is indispensable for viability and regulation of the cell cycle. Recently the general importance of this gene for the eukaryotic cell was shown by the identification of a structural and functional human homologue. The homologous mammalian ALR (Augmenter of Liver Regeneration) genes from man, mouse and rat are involved in the phenomenon of liver regeneration. A low expression rate of the genes is found in all investigated cells and mammalian tissues but it is specifically induced after damage of liver organs and is especially high during spermatogenesis. The alignment of the different proteins identifies a highly conserved carboxy terminus with more than 40% identical amino acids between yeast and mammals. The conserved carboxy terminus is functionally interchangeable between distantly related species like yeast and man. In contrast, the amino terminal parts of the proteins display a high degree of variability and significant differences even among closely related species. This finding leads to the problem whether the amino termini have comparable or divergent functions in different species. In this study we demonstrate by heterologous complementation experiments in yeast that the complete human ALR protein with its own amino terminus is not able to substitute for the yeast scERV1 protein. Fusion proteins of Alrp and scErv1p with the green fluorescence protein were created to investigate the respective subcellular localizations of these homologous proteins in yeast and human cells. In yeast cells human Alrp accumulates in the cytoplasm in contrast to yeast scErv1p that is preferentially associated with yeast mitochondria. Comparable studies with human cells clearly show that the homologous human Alrp is located in the cytosol of these cells. Fractionation experiments and antibody tests with yeast and human mitochondria and cellular extracts verify these findings.
Amino Acid Sequence↗
Augmentation of antitumor immunity by genetically engineered fibroblast cells to express both B7.1 and interleukin-7.
Mouse fibroblasts (H-2(b)) were genetically engineered to express a costimulatory B7.1 and an interleukin-7 (IL-7; Fb/B7.1/IL7). The Fb/B7.1/IL7 cells were then pulsed with an ovalbumin (OVA) epitope (amino acids 257-264, SIINFEKL, H-2 K(b) restricted; Fb/B7. 1/IL7/OVA) and tested for the induction of OVA-specific cytotoxic T lymphocytes (CTLs) in C57BL/6 mice (H-2(b)). The genetically engineered fibroblasts lacking either B7.1 or IL-7 were constructed and used as controls. Immunization with the Fb/B7.1/IL7/OVA cells induced strong cytotoxic activities against OVA-expressing EL4 (EG7) tumor cells. The magnitude of the cytotoxic response in mice with the Fb/B7.1/IL7/OVA cells was significantly higher than the response in mice immunized with any other cell constructs. CD8(+) T cells were a major effector cell-type of antitumor response in the immunized mice with the Fb/B7.1/IL7/OVA cells. Furthermore, immunization with the Fb/B7.1/IL7/OVA cells significantly prolonged the survival period of mice when the mice were injected with EG7 tumor cells one week after the immunization. These results suggest that fibroblasts can be genetically modified to an efficient cell vaccine for the induction of antitumor response.
Adjuvants, Immunologic↗
Recombinant measles viruses expressing heterologous antigens of mumps and simian immunodeficiency viruses.
We have genetically engineered a panel of recombinant measles viruses (rMVs) that express from various positions within the MV genome either the HN or F surface glycoproteins of mumps virus (MuV) or the env, gag or pol proteins from simian immunodeficiency virus (SIV). All rMVs were rescued from the respective antigenomic plasmid constructs; progeny viruses replicated comparably to the progenitor Edmonston B MV, but showed slight propagation retardation, which was dependent on the size and nature of the expressed proteins and on the genomic position of the inserts. All transgenes except that encoding mumps F glycoprotein were faithfully maintained and expressed even after virus amplification by 10(20). Our results suggest possible applications of rMVs as live-attenuated, multivalent vaccines against retroviruses such as SIV and HIV as well as other pathogens more distantly related to MV than MuV.
Animals↗
Vaccine-induced cytotoxic T lymphocytes against human immunodeficiency virus type 1 using two complementary in vitro stimulation strategies.
CD8+ cytotoxic T lymphocytes (CTL) against human immunodeficiency virus type 1 (HIV-1) induced by candidate HIV-1 vaccines may be a mechanism of immune protection against HIV-1 infection. We measured in vitro inducible CD8+ and CD4+ CTL using two in vitro effector cell stimulation strategies. Peripheral blood mononuclear cells (PBMC) for CTL assay were obtained after the third and/or fourth immunization timepoints from 23 healthy, uninfected adult volunteers, of whom 19 received a canarypox virus vaccine expressing HIV-1 env, gag, pol, nef and protease gene products (vCP300) with or without injections of HIV-1(SF-2) rgp120 subunit vaccine and four subjects received only control injections. CD8+ CTL activity was detected employing the two in vitro stimulation strategies against one or more HIV-1 antigens in 15 (79%) of 19 HIV-1 vaccine recipients on at least one occasion and repeatedly against the same antigen in 8 (42%). Canarypox virus-based HIV-1 vaccines represent a step forward in HIV-1 vaccine development.
AIDS Vaccines↗
The role of biotechnologically engineered vaccines and diagnostics in pseudorabies (Aujeszky's disease) eradication strategies.
Modern-day biotechnology has an almost unlimited number of possibilities for reducing the impact of hereditary and infectious diseases. To date one of its most visible and rewarding applications for veterinary medicine has been in the genetic engineering of vaccines and diagnostics to assist in the eventual eradication of pseudorabies (PR, Aujeszky's disease). In the following review we summarize some of the most pertinent issues relative to PR eradication and point out the present and potential role of biotechnology in achieving our goal.
Animals↗
Research advances on transgenic plant vaccines.
In recent years, with the development of genetics molecular biology and plant biotechnology, the vaccination (e.g. genetic engineering subunit vaccine, living vector vaccine, nucleic acid vaccine) programs are taking on a prosperous evolvement. In particular, the technology of the use of transgenic plants to produce human or animal therapeutic vaccines receives increasing attention. Expressing vaccine candidates in vegetables and fruits open up a new avenue for producing oral/edible vaccines. Transgenic plant vaccine disquisitions exhibit a tempting latent exploiting foreground. There are a lot of advantages for transgenic plant vaccines, such as low cost, easiness of storage, and convenient immune-inoculation. Some productions converged in edible tissues, so they can be consumed directly without isolation and purification. Up to now, many transgenic plant vaccine productions have been investigated and developed. In this review, recent advances on plant-derived recombinant protein expression systems, infectious targets, and delivery systems are presented. Some issues of high concern such as biosafety and public health are also discussed. Special attention is given to the prospects and limitations on transgenic plant vaccines.
Administration, Oral↗
Plants for delivery of edible vaccines.
Over the past decade, scientific advances in molecular biology and immunology have improved understanding of many diseases and led to the development of novel strategies for vaccination. The development of plants expressing vaccine antigens is a particularly promising approach. Plant-derived antigenic proteins have delayed or prevented the onset of disease in animals and have proven to be safe and functional in human clinical trials. Future areas of research should further characterize the induction of the mucosal immune system and appropriate crop species for delivery of animal and human vaccines.
Administration, Oral↗