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PubMedDate not supplied

Red fluorescent protein eqFP611 and its genetically engineered dimeric variants.

The red fluorescent protein (FP) eqFP611 from the sea anemone Entacmaea quadricolor shows favorable properties for applications as a molecular marker. Like other anthozoan FPs, it forms tetramers at physiological concentrations. The interactions among the monomers, however, are comparatively weak, as inferred from the dissociation into monomers in the presence of sodium dodecyl sulfate (SDS) or at high dilution. Analysis at the single-molecule level revealed that the monomers are highly fluorescent. For application as fusion markers, monomeric FPs are highly desirable. Therefore, we examine the monomer interfaces in the x-ray structure of eqFP611 to provide a basis for the rational design of monomeric variants. The arrangement of the four beta cans is very similar to that of other green fluorescent protein (GFP-like) proteins such as DsRed and RTMS5. A variety of structural features of the tetrameric interfaces explain the weak subunit interactions in eqFP611. We produce functional dimeric variants by introducing single point mutations in the A/B interface (Thr122Arg, Val124Thr). By contrast, structural manipulations in the A/C interface result in essentially complete loss of fluorescence, suggesting that A/C interfacial interactions play a crucial role in the folding of eqFP611 into its functional form.

Animals↗
PubMed1997

Efficient lysis of CD44v7/8-presenting target cells by genetically engineered cytotoxic T-lymphocytes--a model for immunogene therapy of cervical cancer.

Variant proteins of the CD44 surface glycoprotein family are expressed on many different human tumors and their lymph node metastases. An epitope encoded by sequences of variant exons CD44v7 and v8 and recognized by the monoclonal antibody VFF17 is frequently detected in cervical cancer, whereas the normal cervical epithelium lacks expression of this epitope. We have developed an immunotherapeutic approach for cervical cancer based on the expression of this CD44v7/8 epitope. The single chain antigen-binding fragment of VFF17 was fused to a signal transducing protein (zeta-chain) of the T-cell receptor complex (TCR) and was introduced into a retroviral gene transfer vector. Gene transfer was applied to the murine cytotoxic T-cell line cl96. All recombinant clones expressed the fusion protein on their cell surface. Functionality of the recombinant fusion protein was tested by subjection of several recombinant clones to in vitro cytotoxicity assays. CD44v7/8-expressing target cells were killed efficiently by reprogrammed cl96 in an MHC-independent fashion, whereas CD44v7/ 8-negative cells were not affected. These transfected T cell lines will now be tested in vivo using immune-deficient mice bearing CD44v7/8-expressing tumors.

Amino Acid Sequence↗
PubMed1991

Genetic engineering and overexpression of ribosomal L12 protein genes from three different archaebacteria in E coli.

Genes coding for ribosomal protein L12 from Methanococcus vannielii (Mva), Halobacterium halobium (Hha) and Sulfolobus solfataricus (Sso) have been subcloned in the polylinker region of pUC19. An efficient Shine-Dalgarno sequence has been attached to the 5' end of the genes, and two ochre stop codons have been created at their 3' ends, where necessary. In addition, mutants of the MvaL12 and HhaL12 genes were constructed, which coded for a cysteine residue at the C-terminus of the protein. The constructs were transferred together with the pUC19 polylinker as gene cartridges into different expression vectors. These constructed plasmids were transformed in the appropriate E coli hosts and tested for expression. Two systems were found to work efficiently for overexpression, namely the pKK223-3 vector featuring a tac promoter, and the pT7-5 vector featuring a T7-promoter. The over-expressed proteins were purified to homogeneity; their purity was investigated by one and two-dimensional gel systems, amino acid analysis and N-terminal protein sequencing for 10 steps or more. The amount of protein purified from E coli test cultures bearing the expression plasmids was always more than 2.5 mg/l of medium used.

Amino Acids↗
PubMed1999

Genetic engineering of shikonin biosynthesis hairy root cultures of Lithospermum erythrorhizon transformed with the bacterial ubiC gene.

The biosynthetic pathway to 4-hydroxybenzoate (4HB), a precursor of the naphthoquinone pigment shikonin, was modified in Lithospermum erythrorhizon hairy root cultures by introduction of the bacterial gene ubiC. This gene of Escherichia coli encodes chorismate pyruvate-lyase (CPL), an enzyme that converts chorismate into 4HB and is not normally present in plants. The ubiC gene was fused to the sequence for a chloroplast transit peptide and placed under control of a constitutive plant promoter. This construct was introduced into L. erythrorhizon by Agrobacterium rhizogenes-mediated transformation. The resulting hairy root cultures showed high CPL activity. 4HB produced by the CPL reaction was utilized for shikonin biosynthesis, as shown by in vivo inhibition of the native pathway to 4HB with 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia-lyase. A feeding experiment with [1,7-13C2]shikimate showed that in the absence of AIP the artificially introduced CPL reaction contributed ca. 20% of the overall 4HB biosynthesis in the transgenic cultures. ubiC transformation did not lead to a statistically significant increase of shikonin formation, but to a 5-fold increase of the accumulation of menisdaurin, a nitrile glucoside which is presumably related to aromatic amino acid metabolism.

Cell Line, Transformed↗
PubMed1997

Targeting retroviral vectors to vascular lesions by genetic engineering of the MoMLV gp70 envelope protein.

Targeted gene delivery to vascular lesions is a major challenge in the development of gene therapy protocols for cardiovascular diseases. One approach would be to enable retroviral vectors to accumulate at sites of vascular injury and enhance local vector concentration. An early step in wound repair is the adhesion of platelets to collagen exposed from damaged vasculature. Hence, the Moloney murine leukemia virus (MoMLV) envelope (env) protein was engineered to incorporate a high-affinity collagen-binding domain derived from von Willebrand clotting factor, and expressed in Escherichia coli and in mammalian cells. The chimeric env protein bound tightly to collagen, and virions bearing this collagen-binding env protein exhibited viral titers approaching those of virions expressing wild-type (WT) env protein. The chimeric virions were concentrated on collagen matrices, and they retained their infectivity under conditions in which virions bearing WT env protein were washed away. Targeted delivery of the chimeric env protein to injured mouse aorta and selective binding of the collagen-targeted virions to injured rabbit artery were observed. In comparative studies, vascular smooth muscle cell transduction was demonstrated in catheter-injured carotid arteries following infusion of collagen-targeted virions but not of virions bearing WT env protein. Taken together, these observations demonstrate the ability of collagen-targeted virions to localize gene delivery to sites of vascular injury.

3T3 Cells↗
PubMed2003

Prevention of the initial host immuno-inflammatory response determines the long-term survival of encapsulated myoblasts genetically engineered for erythropoietin delivery.

The present study investigates the respective roles of both the host immune response and the metabolic requirements in determining the long-term survival of erythropoietin-secreting myoblasts within encapsulating polymer membranes. Hollow-fiber capsules loaded with a high density of erythropoietin-secreting C(2)C(12) myoblasts survived poorly in the subcutaneous tissue of syngeneic mice, inducing variable hematocrit responses. To determine the role and the nature of the host response, recipients were treated with anti-inflammatory (diclofenac) and immunosuppressive (dexamethasone, FK506) agents. Only immunosuppressive drugs led to improved graft survival after 5 weeks of implantation, indicating an immune process as the cause of cell death. Furthermore, transient blocking of this process allowed long-term preservation of the implanted cells (> 100 days). The formation of necrotic cell cores inside densely packed devices elicited a local chronic inflammatory reaction. Hence, implants were designed to limit early cell death by inserting a supporting matrix and decreasing the number of loaded cells. The most efficient erythropoietin delivery was obtained with matrix-containing capsules that had received the lowest myoblast density. These results highlight the critical role of initial engraftment in the long-term acceptance of encapsulated myoblasts and the need to limit early cell death in the device to prevent subsequent host immuno-inflammatory responses.

Animals↗
PubMed1998

Genetic engineering of an industrial strain of Saccharopolyspora erythraea for stable expression of the Vitreoscilla haemoglobin gene (vhb).

Several Actinomycetes/Streptomycetes expression vectors are described for expression of the Vitreoscilla haemoglobin gene (vhb) in an industrial erythromycin-producing strain of Saccharopolyspora erythraea. Cloning of vhb under the control of either the thiostrepton-inducible PtipA promoter or the constitutive PermE* promoter led to the production of chemically active haemoglobin (VHb) in Streptomyces lividans TK24 transformed with these constructs. However, theplasmids could not be transformed into Sac. erythraea. Transformants of Sac. erythraea and/or exconjugants were obtained using a novel Escherichia coli/Streptomyces shuttle vector comprised of vhb under the control of the PermE* promoter, the Streptomyces plasmid pIJ350 origin of replication, the thiostrepton-resistance gene (tsr) for selection, and the oriT region which is necessary for conjugal transfer. Increased plasmid stability in Sac. erythraea was obtained by construction of a vector for chromosomal integration. This vector contained the Streptomyces phage phi C31 attachment site for chromosomal integration and vhb expressed under the PmerR promoter and was stably maintained in the chromosome of Sac. erythraea. Shake-flask cultivations of the transformed Sac. erythraea strain with the chromosomally integrated vhb gene show that vhb is expressed in an active form. The corresponding amount of erythromycin produced in the vhb-expressing strain was approximately 60% higher relative to the original VHb-negative strain.

Bacterial Proteins↗
PubMed2005

Enhanced inhibition of syngeneic murine tumors by combinatorial therapy with genetically engineered HSV-1 expressing CCL2 and IL-12.

Herpes simplex viruses type 1 (HSV-1) that lack the gamma(1)34.5 gene are unable to replicate in the central nervous system (CNS), but maintain replication competence in actively dividing tumors. To determine if antitumor therapy by M002, a gamma(1)34.5(-) HSV that expresses interleukin-12 (IL-12), could be augmented by combinatorial therapy with another gamma(1)34.5-deleted HSV-1 engineered to express the chemokine CCL2, Neuro-2a tumors were established subcutaneously in the syngeneic A/J mouse strain. Tumors received multiple injections intratumorally either of saline, the parent, non-cytokine-expressing virus R3659, M002, M010 (gamma(1)34.5(-) HSV expressing CCL2), or a combination of M002 and M010. Efficacies were evaluated by monitoring inhibition of tumor growth over time. Results demonstrated the following: (1) inhibition of tumor growth was most pronounced in tumors treated with a combination of M002 and M010; (2) enhanced tumor growth inhibition for the combinatorial treatment group was statistically significant compared to either M002 or M010 alone; and (3) the variability between slopes of the tumor growth rates within an individual treatment group appeared to be virus-dependent, and was reproducible between experiments. Our results demonstrate that combinatorial cytokine/chemokine gamma(1)34.5(-) HSV therapies can provide superior antitumor effects in experimental tumors as a model for malignancies arising in the brain.

Animals↗
PubMed2002

Genetic engineering of the glucocorticoid receptor by fusion with the herpes viral protein VP22 causes selective loss of transactivation.

The development of methods for engineering proteins with novel properties opens the way to manipulating intracellular processes in a therapeutically useful way. Glucocorticoids, acting via glucocorticoid receptors (GR), are potent anti-inflammatory agents, acting to oppose nuclear factor kappa B (NF kappa B) function. The herpes viral protein, VP22, has been reported to confer intercellular trafficking activity on 'cargo' proteins, potentially facilitating gene therapy with intracellular proteins. VP22GR, resulting from the addition of VP22 to the N terminal of GR, was equipotent with the wild-type GR in opposing NF kappa B p65-driven expression of an NF kappa B reporter gene. Surprisingly, VP22GR was incapable of inducing transactivation of positive glucocorticoid reporter genes (MMTV-luc and TAT3-luc). Furthermore, the VP22GR had powerful dominant negative activity on both endogenous and exogenous GR transactivation. VP22GR was cytoplasmic in quiescent cells, and after hormone addition underwent nuclear translocation to share the same distribution as the GR. The ability of the VP22GR to selectively confer and enhance glucocorticoid-dependent transrepression of NF kappa B may be of use therapeutically in e.g. transplant rejection, inflammatory arthritis or asthma.

Animals↗
PubMed1988

Genetic engineering of Schizosaccharomyces pombe: a system for gene disruption and replacement using the ura4 gene as a selectable marker.

A system is described for gene disruption and replacement in Schizosaccharomyces pombe based on the homologous selectable marker, ura4, the structural gene for orotidine-5'-phosphate decarboxylase. The presence of a single copy of the wild-type gene can rescue a ura4 auxotrophic mutant. Furthermore, ura4- cells can be selected for in the presence of 5-fluoroorotic acid (5-FOA). This allows a convenient means of selecting for both forward and backward mutations. The sequence of a 1.8 kb HindIII fragment which contains the functional gene is reported. It encodes a single open reading frame of 264 amino acids which shows considerable conservation with the orotidine-5'-phosphate (OMP) decarboxylases from other organisms. The ura4 transcript is approximately 850 nucleotides long. It begins 51 bp upstream of the protein coding sequence and is unusual in that transcription termination occurs at or very close to the translational stop codon. To facilitate the use of ura4 in gene disruption experiments we have also constructed a novel strain of S. pombe called ura4-D18, in which the 1.8 kb HindIII fragment has been deleted from the chromosome. Using a combination of this strain and vectors containing ura4 as a selectable marker, we present a general method for targeting recombination events to the chromosomal locus under investigation.

Amino Acid Sequence↗
PubMed1995

Fibroblasts genetically engineered to secrete cytokines suppress tumor growth and induce antitumor immunity to a murine glioma in vivo.

The genes for interleukin (IL)-2, interferon (IFN)-gamma, or both IL-2 and IFN-gamma were introduced into a mouse fibroblast cell line (LM) expressing defined major histocompatibility complex determinants (H-2k). The cytokine-secreting cells were then co-transplanted with the Gl261 murine glioma cell line (H-2b) into syngeneic C57BL/6 mice that differed at the major histocompatibility complex from the cytokine-secreting cells. The period of survival of mice with glioma treated with IL-2- or IL-2/IFN-gamma-secreting allogeneic cells was significantly prolonged (P < 0.025) relative to the survival of mice receiving equivalent numbers of tumor cells alone or mice with glioma treated with nonsecreting fibroblast (LM) cells. Gliomas in the treated mice had an extensive lymphocytic cell infiltrate. Using a 51Cr release assay, the specific release of isotope from labeled Gl261 cells co-incubated with spleen from mice injected with the glioma cells and IL-2-secreting fibroblasts was higher (P < 0.001) than the release from glioma cells co-incubated with spleen cells from nonimmunized mice. Significantly higher levels of release (P < 0.005) were found in the group immunized with fibroblasts secreting both IL-2 and IFN-gamma. Based upon the effect of monoclonal antibodies for T-cell subsets on the antiglioma response, the immunity was mediated predominantly by natural killer/lymphokine-activated killer cells.

Animals↗
PubMed2000

Genetic engineering of Escherichia coli to produce a 1:1 complex of the anabaena sp. PCC 7120 nuclease NucA and its inhibitor NuiA.

A series of T7-promoter based bicistronic expression vectors was constructed in order to produce the complex of the Anabaena sp. PCC 7120 DNA/RNA non-specific nuclease NucA and its inhibitor NuiA. With all constructs, tandem expression of nucA and nuiA results in aggregation and inclusion body formation of NucA, independent of the order of the genes, the relative expression of the two proteins and the temperature applied during expression. Two constructs in which nuiA is the first and nucA the second cistron lead to an approximately one order of magnitude higher expression of nuiA compared with nucA. In these cells inclusion bodies are formed which contain NucA and NuiA in a 1:1 molar ratio. The complex can be solubilized with 6M urea after disruption of the cells by sonication, renatured by dialysis and purified to homogeneity. 2mg of the complex are obtained from 1l Escherichia coli culture. As shown by gel filtration and analytical ultracentrifugation, our system leads to a highly pure and homogeneous complex preparation, as required for biophysical and structural studies. Thus, our new method is a superior alternative for the production of the NucA/NuiA complex in which separately produced nuclease and inhibitor are mixed, and an excess of one or the other component, as well as aggregates of NucA, have to be removed from the preparation.

Algal Proteins↗
PubMed1992

Insecticidal properties of genetically engineered baculoviruses expressing an insect juvenile hormone esterase gene.

Exploring the possibility of enhancing the properties of baculoviruses as biological control agents of insect pests, we tested the effect of expressing an insect gene (jhe) encoding juvenile hormone esterase. Juvenile hormone esterase inactivates juvenile hormone, which regulates the outcome of an insect molt. A cDNA encoding the juvenile hormone esterase of Heliothis virescens was inserted into the genome of Autographa californica nuclear polyhedrosis virus such that the gene was expressed under the control of a strong, modified viral promoter. This virus, however, naturally encodes an ecdysteroid UDP-glucosyltransferase which inactivates ecdysone, the hormone which initiates molting. Since ecdysteroid UDP-glucosyltransferase could mask the effects of jhe expression by blocking molting entirely, jhe-expressing viruses in which the ecdysteroid UDP-glucosyltransferase gene was deleted or disrupted were constructed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of proteins from infected cells revealed several intracellular proteins and two major secreted proteins which reacted with antibodies to authentic juvenile hormone esterase. Western blot analysis coupled with tunicamycin treatment indicated that differential glycosylation was responsible for the multiple products. Hemolymph of recombinant virus-infected fourth-instar Trichoplusia ni larvae contained levels of juvenile hormone esterase activity 40-fold higher than maximal levels found in uninfected larvae. However, little or no difference in developmental characteristics, weight gain, or time of mortality was observed between insects infected with the jhe-expressing viruses and control viruses.

Animals↗
PubMed1991

Optimization of cell culture conditions for G-CSF (granulocyte-colony stimulating factor) production by genetically engineered Namalwa KJM-1 cells.

An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 micrograms/ml/day using a 25 cm2 tissue culture flask, even though the cell number was above 7 x 10(5) cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, Biofermenter (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 4 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1 x 10(7) cells/ml), and the amount of G-CSF reached 41 micrograms/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.

Base Sequence↗
PubMed2005

Continuous, noninvasive monitoring of local microscopic inflammation using a genetically engineered cell-based biosensor.

Using an inflammation-responsive regulatory element as a molecular sensor, we established a cell-based biosensor for continuous, noninvasive monitoring of local microscopic inflammation in vivo. Glomerular mesangial cells were stably transfected with a marker gene encoding secreted alkaline phosphatase (SEAP) under the control of the kappaB enhancer elements. The established cells secreted SEAP in vitro in response to proinflammatory cytokines as well as to soluble factors produced by inflamed glomeruli. To examine feasibility of using the established cells for in vivo monitoring of local microscopic inflammation, the sensor cells were transferred selectively into rat glomeruli via the renal circulation. After induction of acute glomerulonephritis, the serum level of SEAP was increased transiently in cell-transferred nephritic rats. The kinetics of serum SEAP was closely correlated with the natural course of the inflammation, and the increase in SEAP was attenuated by suppression of inflammation using an immunosuppressive drug, cyclophosphamide. Neither cell-transferred normal rats nor nephritic rats without cell transfer exhibited increase in the serum level of SEAP. When the sensor cells were transferred extrarenally, elevation of serum SEAP was not observed in nephritic rats, confirming that the locally settled sensor cells responded only to local inflammation. These results suggested that, without invasive procedures like tissue biopsies, continuous monitoring of microscopic inflammation is feasible in vivo via locally created, cell-based biosensors.

Acute Disease↗
PubMed2003

Induction of an antitumor immunological response by an intratumoral injection of dendritic cells pulsed with genetically engineered Semliki Forest virus to produce interleukin-18 combined with the systemic administration of interleukin-12.

OBJECT: The aim of this study was to investigate further immunogene treatment of malignant brain tumor to improve its therapeutic efficacy. METHODS: Intratumoral dendritic cells pulsed with Semliki Forest virus (SFV)-interleukin-18 (IL-18) and/or systemic IL-12 were injected into mice bearing the B16 brain tumor. To study the immune mechanisms involved in tumor regression, we monitored the growth of implanted B16 brain tumor cells in T cell-depleted mice and IFNgamma-neutralized mice. To analyze the protective immunity created by tumor inoculation, B16 cells were injected into the left thighs of mice that had received an inoculation, and tumor growth was monitored. The local delivery of dendritic cells pulsed with IL-18 bound by SFV combined with the systemic administration of IL-12 enhanced the induction of the T helper type 1 response from tumor-specific CD4+ and CD8+ T cells and natural killer cells as well as antitumor immunity. Interferon-gamma is partly responsible for this IL-18-mediated antitumor immunity. Furthermore, the protective immunity is mediated mainly by CD8+ T cells. CONCLUSIONS: Immunogene therapy that combines the local administration of dendritic cells pulsed with IL-18 bound by SFV and the systemic administration of IL-12 may be an excellent candidate for the development of a new treatment protocol. A self-replicating SFV system may therefore open a novel approach for the treatment of malignant brain tumor.

Animals↗
PubMed1996

Genetically engineered particulate virus-like structures and their use as vaccine delivery systems.

The Orbivirus genus within the family Reoviridae consists of nonenveloped architecturally complex viruses. The icosahedral viruses are 810 A diameter in size and are comprised of two protein shells containing seven proteins (VP1-VP7), surrounding a genome of ten double-stranded RNA segments. The prototype virus, bluetongue virus (BTV), is the etiological agent of a disease that can reach epidemic proportions among sheep and cattle. To develop highly protective virus-like particles, we have developed novel baculovirus multigene expression vector systems which have allowed us to coexpress three, four or five BTV genes from single recombinant vectors. The resultant particulate structures resemble BTV virus-like and subvirus-like particles which are structurally and immunologically indistinguishable from the BTV, and preliminary clinical trials have verified this vaccines safety and efficacy. Unlike live virus vaccines, VLPs are noninfectious and lack virus (or other) DNA/RNA required for replication. VLPs do not replicate in host cells. However, sheep trials have shown that VLPs are more immunogenic than subunit vaccines (viral proteins), or viruses killed by chemical inactivation. In addition, they are effective at eliciting humoral, cell-mediated and mucosal immunities. Virus-like particles (VLPs) are safe to produce and handle. The baculovirus vector and host cells used to make VLPs do not come from mammalian sources (hence they do not contain mammalian-derived pathogens). The multicomponent VLPs have also been utilized as vaccine delivery systems for multiple immunogens including B and T cell epitopes. The expression system described here is a tool which may have a range of applications in industries employing biotechnology to produce vaccines, insecticides, diagnostic and protein reagents.

Animals↗