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PubMed1999

Genetic engineering of dendritic cells to express immunosuppressive molecules (viral IL-10, TGF-beta, and CTLA4Ig).

There is growing evidence that, in addition to their role as initiators of immune responses, dendritic cells (DC) can exhibit tolerogenic properties. Immature DC deficient in cell surface costimulatory/accessory molecules can prolong organ and pancreatic islet allograft survival, whereas in vitro manipulation of DC by exposure to a variety of factors (e.g., viral interleukin-10; CTLA4Ig) can confer tolerogenic properties on these cells. Genetic engineering of DC to express immunosuppressive molecules is, in theory, an attractive approach to the therapy of allograft rejection and possibly, autoimmune disorders.

Abatacept↗

PubMed2003

Brain transplantation of genetically engineered human neural stem cells globally corrects brain lesions in the mucopolysaccharidosis type VII mouse.

In the present study, we investigated the feasibility of using human neural stem cells (NSCs) in the treatment of diffuse central nervous system (CNS) alterations in a murine model of mucopolysaccharidosis VII (MPS VII), a lysosomal storage disease caused by a genetic defect in the beta-glucuronidase gene. An immortalized NSC line derived from human fetal telencephalon was genetically engineered to overexpress beta-glucuronidase and transplanted into the cerebral ventricles of neonatal MPS VII mouse. Transplanted human NSCs were found to integrate and migrate in the host brain and to produce large amount of beta-glucuronidase. Brain contents of the substrates of beta-glucuronidase were reduced to nearly normal levels, and widespread clearing of lysosomal storage was observed in the MPS VII mouse brain at 25 days posttransplantation. The number of engrafted cells decreased markedly after the transplantation, and it appears that the major cause of the cell death was not the immune response of the host but apoptotic cell death of grafted human NSCs. Results showed that human NSCs would serve as a useful gene transfer vehicle for the treatment of diffuse CNS lesions in human lysosomal storage diseases and are potentially applicable in the treatment of patients suffering from neurological disorders.

Animals↗

PubMed2006

Integration of genetically modified adult astrocytes into the lesioned rat spinal cord.

Combination of ex vivo gene transfer and cell transplantation is now considered as a potentially useful strategy for the treatment of spinal cord injury. In a perspective of clinical application, autologous transplantation could be an option of choice. We analyzed the fate of adult rat cortical astrocytes genetically engineered with a lentiviral vector transplanted into a lesioned rat spinal cord. Cultures of adult rat cortical astrocytes were infected with an HIV-1-derived vector (TRIP-CMV-GFP) and labeled with the fluorescent dye Hoechst. Transfected and labeled astrocyte suspension was injected at T11 in rats in which spinal cord transection at T7-T8 levels had been carried out 1 week earlier. Six weeks after grafting, the animals were sacrificed and transplants were retrieved either by Hoechst fluorescence or by immunohistochemistry for detection of glial fibrillary acidic protein (GFAP) and vimentin. Grafted astrocytes expressing green fluorescent protein (GFP) were found both at the injection and transection sites. Genetically modified astrocytes thus survived, integrated, and migrated within the host parenchyma when grafted into the completely transected rat spinal cord. In addition, they retained some ability to express the GFP transgene for at least 6 weeks after transplantation. Adult astrocytes infected with lentiviral vectors can therefore be a valuable tool for the delivery of therapeutic factors into the lesioned spinal cord.

Animals↗

PubMed2001

Differential cellular compartmentalization of the nuclear receptor SpSHR2 splicing variants in early sea urchin embryos.

SpSHR2 is a member of the nuclear receptor superfamily, expressed in embryos, larvae, and adult tissues of sea urchin. During embryonic development, two receptor isoforms are produced via alternative splicing. One exhibits the typical structure of nuclear receptors (SpSHR2-full length), whereas the other is missing the entire LBD (SpSHR2-splice variant). DNA-constructs encoding these isoforms and two additional in vitro generated deletion mutants were engineered in an expression vector carrying the myc-tag. Expression of the tagged isoforms in S. purpuratus embryos showed that the exogenous SpSHR2 full-length protein displays a similar subcellular localization as the endogenous receptor. In early cleavage stages (4-cells), the full-length isoform is predominantly localized in the nucleus, whereas two cell divisions later (16-cells) protein accumulations are detected in both the nucleus and cytoplasm. To the contrary, the SpSHR2-splice variant is confined in the embryonic nuclei both at 4- and 16-cell stage embryos. Analysis of the intracellular distribution of two receptor mutants, one having a deletion within the DBD (DeltaP) and the other a truncation of the C-terminal F-domain (DeltaF), revealed that DeltaP is localized similarly to full-length receptor, whereas DeltaF is maintained in the nucleus, similar to the SpSHR2 splice variant. Investigation of the DNA binding and dimerization properties of the two SpSHR2 isoforms demonstrated that they recognize and bind to a DR1-element as monomers, whereas DeltaP does not bind DNA and DeltaF binds to DR1 poorly. These results suggest that the receptor's putative LBD is responsible for the differential subcellular localization of the two natural SpSHR2-isoforms in early development.

Active Transport, Cell Nucleus↗

PubMed2002

Production of patchouli mild mosaic virus resistant patchouli plants by genetic engineering of coat protein precursor gene.

Patchouli (Pogostemon cablin (Blanco) Benth), an aromatic crop which yields an essential oil, is widely cultivated in South-east Asia. Patchouli mild mosaic virus (PaMMV) infects patchouli plants and causes decrease in leaf biomass and essential oil yield. Transgenic patchouli plants with PaMMV coat protein precursor (CP-P) gene have been produced by Agrobacterium-mediated transformation. PaMMV CP-P gene integration into the patchouli genome was confirmed by the PCR method and by Southern blot analysis. The transformants were estimated to contain one to three copy genes using Southern blot analysis. The transformant with three copy genes was tested for the resistance to PaMMV by artificially inoculating plants grown in an environmentally controlled cabinet, and this transformant was found to be highly resistant to PaMMV. The transgenic patchouli plant with PaMMV CP-P gene should provide valuable material for protecting against PaMMV.

Blotting, Southern↗

PubMed1994

Expression of modified human cytochrome P450 1A1 in Escherichia coli: effects of 5' substitution, stabilization, purification, spectral characterization, and catalytic properties.

Human cytochrome P450 (P450) 1A1 is primarily an extrahepatic enzyme and is important because of its roles in the activation of polycyclic hydrocarbons and other xenobiotic chemicals. Purification of active enzyme from human tissues has not been successful. We report the expression and purification of the recombinant enzyme from Escherichia coli. A full-length cDNA of human cytochrome P450 1A1 and several modified constructs were engineered into a pCW vector and used to transform E. coli cells. Little expression was observed with the native sequence and several modified constructs, but successful expression (20-25 nmol membrane-bound P450 1A1 per liter of culture) was achieved with a construct in which the Ala codon GCT was placed in the second position and the 5'-terminal codons were maximized for AT content and minimized for the potential of secondary structure formation of the mRNA transcript. alpha-Naphthoflavone was found to protect against denaturation by detergents during solubilization and was added to buffers used for purification. The recombinant P450 1A1 was purified to electrophoretic homogeneity after two ion-exchange chromatography steps in approximately 50% yield. N-Terminal amino acid sequence analysis verified the expected first 21 residues, with the exception of the terminal Met. The isolated human ferric P450 1A1 was predominantly in the high spin state, in contrast to the orthologous rat and rabbit enzymes. Recombinant P450 1A1 catalyzed 7-ethoxyresorufin O-deethylation and benzo[a]pyrene 3-hydroxylation with Km values of 0.58 and 15 microM and Vmax values of 8.3 and 2.5 nmol min-1 (nmol P450 1A1)-1, respectively. The successful expression and purification of human P450 1A1 should increase the availability of this enzyme and the generation of antibodies for further biochemical and other biological studies.

Amino Acid Sequence↗

PubMed2000

Efficient production of recombinant brazzein, a small, heat-stable, sweet-tasting protein of plant origin.

Brazzein is a 54-amino-acid sweet-tasting protein first isolated from the fruit of Pentadiplandra brazzeana Baillon found in West Africa. Brazzein, as isolated from the fruit, is 500 times sweeter than sucrose on a weight basis (9500 times sweeter on a per-molecule basis). A minor component of brazzein from fruit, des-pGlu1-brazzein, has 53 amino acid residues and has twice the sweetness of the parent protein. We have designed a gene for des-pGlu1- brazzein that incorporates codons that are optimal for protein production in Escherichia coli. Production of brazzein from the chemically synthesized gene resulted in recombinant protein with sweetness similar to that of brazzein isolated from the original source. The best yields were achieved by producing brazzein as a fusion with staphylococcal nuclease with a designed cyanogen bromide cleavage site. Because of its intense sweetness and stability at high pH and temperature, brazzein is an ideal system for investigating the chemical and structural requirements involved in sweet-taste properties. This efficient protein production system for brazzein will facilitate such investigations.

Base Sequence↗

PubMed1998

Isolation and expression analysis of peanut chlorotic streak caulimovirus (PClSV) full-length transcript (FLt) promoter in transgenic plants.

A promoter fragment from peanut chlorotic streak caulimovirus (PClSV) full-length transcript (FLt) was identified and later modified to have duplicated enhancer domain. The FLt promoter with its single or double enhancer domains, fused with the GUS reporter gene to form chimeric gene constructs, showed a high level of expression of these genes in cells and transgenic plants. The FLt promoter with its double enhancer domain gives an average threefold greater expression of genes compared to the FLt promoter with its single enhancer domain in transgenic plants. In young seedlings the expression was in the order root > leaf > stem. The histochemical GUS assay in young seedlings showed more activity in root tips and leaf midribs, veins, and other vascular tissues. The expression from the PClSV FLt promoter was compared with that from the figwort mosaic virus promoter in transgenic plants. These constitutive promoters were comparable in respect to GUS expression level.

Caulimovirus↗

PubMed1995

Effect of a cecropin-like synthetic peptide (Shiva-3) on the sporogonic development of Plasmodium berghei.

The effect of a synthetic cecropin-like peptide, Shiva-3, on in vitro ookinete development and on the early sporogonic stages of Plasmodium berghei in the midgut of Anopheles albimanus was investigated. Peptide concentrations of 75 and 100 microM were effective (P < 0.05) in reducing ookinete production and the number of infected mosquitoes in almost all experiments. These peptide concentrations in the midgut were not toxic for the survival of the mosquitoes. Complete inhibition was obtained if 100 microM Shiva-3 was applied in the first 8 hr of parasite development. The deleterious effect of the peptide on the parasite was effective after exposure for as short as 50 sec and the permanence of free peptide in the mosquito midgut was estimated to be of a minimum of 5 min. These observations indicate the possibility of using Shiva-like peptide genes to engineering malaria-resistant vectors as an alternative in malaria control strategies.

Animals↗

PubMed2000

Dual renin gene targeting by Cre-mediated interchromosomal recombination.

This study describes a new approach to targeting clustered genes. Our study began with the establishment of two lines of mice carrying different mutations in either Ren1 or Ren2. These two genes, both encoding renin, span over 40 kb in tandem on chromosome 1. Each gene was mutated by gene targeting to contain loxP sites. These two mutants and Cre transgenic mice were mated to produce offspring carrying the mutant Ren1 and Ren2 genes, as well as the Cre transgene concurrently. Initially, two mutant Ren genes were located on separate chromosomes. Southern analysis of mice from the second generation revealed that the mutant Ren1 and Ren2 were interchromosomally recombined at the loxP sites to produce a new dually mutated allele on the chromosome at the rate of 9.6% (7/73). Thus, interchromosomal recombination can be efficiently programmed by mating as designed using the Cre-loxP system.

Animals↗

PubMed1998

Genetically engineered vaccines: an overview.

Despite the early success demonstrated with the hepatitis B vaccine, no other recombinant engineered vaccine has been approved for use in humans. It is unlikely that a recombinant vaccine will be developed to replace an existing licensed human vaccine with a proven record of safety and efficacy. This is due to the economic reality of making vaccines for human use. Genetically engineered subunit vaccines are more costly to manufacture than conventional vaccines, since the antigen must be purified to a higher standard than was demanded of older, conventional vaccines. Each vaccine must also be subjected to extensive testing and review by the FDA, as it would be considered a new product. This is costly to a company in terms of both time and money and is unnecessary if a licensed product is already on the market. Although recombinant subunit vaccines hold great promise, they do present some potential limitations. In addition to being less reactogenic, recombinant subunit vaccines have a tendency to be less immunogenic than their conventional counterparts. This can be attributed to these vaccines being held to a higher degree of purity than was traditionally done for an earlier generation of licensed subunit vaccines. Ironically, the contaminants often found in conventional subunit vaccines may have aided in the inflammatory process, which is essential for initiating a vigorous immune response. This potential problem may be overcome by employing one of the many new types of adjuvants that are becoming available for use in humans. Recombinant subunit vaccines may also suffer from being too well-defined, because they are composed of a single antigen. In contrast, conventional vaccines contain trace amounts of other antigens that may aid in conferring an immunity to infectious agents that is more solid than could be provided by a monovalent vaccine. This problem can be minimized, where necessary, by creating recombinant vaccines that are composed of multiple antigens from the same pathogen. These issues are less of a concern with a live attenuated vaccine, since these vaccines are less costly, require fewer steps to manufacture, and elicit long-lived immunity after only a single dose. Unfortunately, live vaccines carry a higher risk of vaccine-induced complications in recipients that make their use in highly developed, litiginous countries unlikely. In lesser developed countries, where the prevalence of disease and the need for effective vaccines outweighs the risk associated with their administration, live vaccines may play an important role in human health. This review has attempted to make the reader aware of some of the current approaches and issues that are associated with the development of these vaccines. Genetically engineered vaccines hold great promise for the future, but the potential of these vaccines to improve human and animal health has yet to be fully realized.

Animals↗

PubMed1995

The complete nucleotide sequence of RNA 3 of a peach isolate of Prunus necrotic ringspot virus.

The complete nucleotide sequence of RNA 3 of the PE-5 peach isolate of Prunus necrotic ringspot ilarvirus (PNRSV) was obtained from cloned cDNA. The RNA sequence is 1941 nucleotides and contains two open reading frames (ORFs). ORF 1 consisted of 284 amino acids with a calculated molecular weight of 31,729 Da and ORF 2 contained 224 amino acids with a calculated molecular weight of 25,018 Da. ORF 2 corresponds to the coat protein gene. Expression of ORF 2 engineered into a pTrcHis vector in Escherichia coli results in a fusion polypeptide of approximately 28 kDa which cross-reacts with PNRSV polyclonal antiserum. Analysis of the coat protein amino acid sequence reveals a putative "zinc-finger" domain at the amino-terminal portion of the protein. Two tetranucleotide AUGC motifs occur in the 3'-UTR of the RNA and may function in coat protein binding and genome activation. ORF 1 homologies to other ilarviruses and alfalfa mosaic virus are confined to limited regions of conserved amino acids. The translated amino acid sequence of the coat protein gene shows 92% similarity to one isolate of apple mosaic virus, a closely related member of the ilarvirus group of plant viruses, but only 66% similarity to the amino acid sequence of the coat protein gene of a second isolate. These relationships are also reflected at the nucleotide sequence level. These results in one instance confirm the close similarities observed at the biophysical and serological levels between these two viruses, but on the other hand call into question the nomenclature used to describe these viruses.

Amino Acid Sequence↗

PubMed1999

Engineering of noninfectious HIV-1-like particles containing mutant gp41 glycoproteins as vaccine candidates that allow vaccinees to be distinguished from HIV-1 infectees.

Many AIDS vaccine candidates under development may elicit immune responses similar to those observed in and used to screen human immunodeficiency virus type 1 (HIV-1)-infected individuals. Therefore, it is important to develop vaccine candidates that incorporate antigenic markers and allow vaccinees to be distinguished from HIV-1 infectees. To this end, we introduced a series of mutations into and in the vicinity of the major immunodominant region (MIR) of gp41 (residues 598-609), a domain recognized by almost all HIV-1 infectees, and evaluated whether HIV-1-like particles incorporating such mutant glycoproteins could be expressed in mammalian cells. Results indicated that although up to three consecutive amino acids could be replaced within MIR without significantly affecting particle formation or gp160 processing, deletions within MIR impaired envelope processing. Replacement of HIV-1 MIR by part or most of the corresponding domain from other lentiviruses markedly decreased or abolished gp160 processing. Synthetic peptides corresponding to a mutated MIR incorporating three amino acid replacements were not recognized by a panel of sera from HIV-1 infectees, suggesting that HIV-1-like particles with this type of mutation represent potential candidate vaccines that could allow vaccinees to be distinguished from HIV-1 infectees.

AIDS Vaccines↗

PubMed2000

Intranasal immunization with mumps virus DNA vaccine delivered by influenza virosomes elicits mucosal and systemic immunity.

To improve the efficiency of liposome-mediated DNA transfer as a tool for gene therapy or vaccinology, we have further developed a new delivery system based on the modified immunopotentiating reconstituted influenza virus (IRIV). In this study, we engineered a plasmid DNA vector expressing the mumps virus hemagglutinin or the fusion protein. The administration of this DNA vaccine delivered by influenza virosomes, in combination with the mucosal adjuvant Escheriagen via the intranasal route, was efficient for inducing an immune response, both mucosally and systemically, in mice. The production of IgG2a mumps virus-specific antibodies and the secretion of interleukin 10 (IL-10) by antigen-specific T cells indicated that not only Th1 but also Th2 responses were induced by this DNA vaccine formulation. These results suggest that cationic virosomes in combination with Escheriagen may have great potential as an efficient delivery system for intranasal DNA immunization and provide an immune barrier at the mucosal sites.

Administration, Intranasal↗

PubMed2001