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At least 487 records · Page 27Linked to original sources
PubMed2002

Modulation of adenovirus vector tropism via incorporation of polypeptide ligands into the fiber protein.

The efficacy of adenovirus (Ad)-based gene therapy might be significantly improved if viral vectors capable of tissue-specific gene delivery could be developed. Previous attempts to genetically modify the tropism of Ad vectors have been only partially successful, largely due to the limited repertoire of ligands that can be incorporated into the Ad capsid. Early studies identified stringent size limitations imposed by the structure of the Ad fiber protein on ligands incorporated into its carboxy terminus and thus limited the range of potential ligand candidates to short peptides. We have previously identified the HI loop of the fiber knob domain as a preferred site for the incorporation of targeting ligands and hypothesized that the structural properties of this loop would allow for the insertion of a wide variety of ligands, including large polypeptide molecules. In the present study we have tested this hypothesis by deriving a family of Ad vectors whose fibers contain polypeptide inserts of incrementally increasing lengths. By assessing the levels of productivity and infectivity and the receptor specificities of the resultant viruses, we show that polypeptide sequences exceeding by 50% the size of the knob domain can be incorporated into the fiber with only marginal negative consequences on these key properties of the vectors. Our study has also revealed a negative correlation between the size of the ligand used for vector modification and the infectivity and yield of the resultant virus, thereby predicting the limits beyond which further enlargement of the fiber knob would not be compatible with the virion's integrity.

Adenoviruses, Human↗
PubMed2001

Engineering zucchini yellow mosaic potyvirus as a non-pathogenic vector for expression of heterologous proteins in cucurbits.

Plant virus vectors provide an attractive biotechnological tool for the transient expression of foreign genes in whole plants. As yet there has been no use of recombinant viruses for the improvement of commercial crops. This is mainly because the viruses used to create vectors usually cause significant yield loss and can be transmitted in the field. A novel attenuated zucchini yellow mosaic potyvirus (AG) was used for the development of an environmentally safe non-pathogenic virus vector. The suitability of AG as an expression vector in plants was tested by analysis of two infectious viral constructs, each containing a distinct gene insertion site. Introduction of a foreign viral coat protein gene into AG genome between the P1 and HC-Pro genes, resulted in no expression in planta. In contrast, the same gene was stably expressed when inserted between NIb and CP genes, suggesting that this site is more suitable for a gene vector. Virus-mediated expression of reporter genes was observed in squash and cucumber leaves, stems, roots and edible fruit. Furthermore, AG stably expressed human interferon-alpha 2, an important human anti-viral drug, without affecting plant development and yield. Interferon biological activity was measured in cucumber and squash fruit. Together, these data corroborate a biotechnological utility of AG as a non-pathogenic vector for the expression of a foreign gene, as a benefit trait, in cucurbits and their edible fruit.

Amino Acid Sequence↗
PubMed2002

Design of an HIV-1 lentiviral-based gene-trap vector to detect developmentally regulated genes in mammalian cells.

The recent development of HIV-1 lentiviral vectors is especially useful for gene transfer because they achieve efficient integration into nondividing cell genomes and successful long-term expression of the transgene. These attributes make the vector useful for gene delivery, mutagenesis, and other applications in mammalian systems. Here we describe two HIV-1-based lentiviral vector derivatives, pZR-1 and pZR-2, that can be used in gene-trap experiments in mammalian cells in vitro and in vivo. Each lentiviral gene-trap vector contains a reporter gene, either beta-lactamase or enhanced green fluorescent protein (EGFP), that is inserted into the U3 region of the 3' long terminal repeat. Both of the trap vectors readily integrate into the host genome by using a convenient infection technique. Appropriate insertion of the vector into genes causes EGFP or beta-lactamase expression. This technique should facilitate the rapid enrichment and cloning of the trapped cells and provides an opportunity to select subpopulations of trapped cells based on the subcellular localization of reporter genes. Our findings suggest that the reporter gene is driven by an upstream, cell-specific promoter during cell culture and cell differentiation, which further supports the usefulness of lentivirus-based gene-trap vectors. Lentiviral gene-trap vectors appear to offer a wealth of possibilities for the study of cell differentiation and lineage commitment, as well as for the discovery of new genes.

Animals↗
PubMed1999

Replacement vectors for localized gene cloning in the specified region of Streptomyces lividans 66 and model method for the screening of desired recombinants via counter-selection.

Two DNA fragments (3.5 kb and 3.8 kb in size) flanking both ends of the phi HAU3R gene in the genome of Streptomyces lividans 66 were determined and cloned in their natural relative orientations in pIJ653, a cosmid vector derived from the multi-copy Streptomyces plasmid pIJ101, resulting in pHZ806. After insertion of spectinomycin/streptomycin (spc/str) resistance gene into the pIJ101 replication region in pHZ806 and insertion of a hygromycin (hyg) resistance gene between 3.5 kb and 3.8 kb DNA fragments, a new vector with a non-functional Streptomyces replicon, pHZ808, was obtained. In principle, any DNA fragment cloned between 3.5 kb and 3.8 kb fragments of this vector can be stably integrated between the two corresponding regions after introduction into the wild-type S.lividans strains, with synchronous replacement of the DNA between the two regions of the chromosome within which phi HAU3R gene is located. The resultant recombinant strains will thus become phi HAU3-sensitive (phi HAU3S). This phenotype could serve as a good indication that the desired gene replacement has occurred. This principle was demonstrated to be successful using pHZ808 as vector. phi HAU3-resistance gene (phi HAU3R) from the genome of S.lividans 66 was substituted by the hygromycin resistance gene (hyg) from pHZ808. An additional advantage of using pHZ808 as the vector to clone foreign genes is that hyg could be served as a reportor gene to imply that foreign DNA fragment has been co-integrated with hyg. The recombinants will have both hygR and phi HAU3S.

Anti-Bacterial Agents↗
PubMed1998

High efficiency gene transfer to the central nervous system of rodents and primates using herpes virus vectors lacking functional ICP27 and ICP34.5.

The safe and efficient use of herpes simplex virus (HSV)-based vectors to deliver genes of potentially therapeutic benefit to the central nervous system will require their effective disablement by the inactivation of viral genes required for lytic growth. Here we report that viruses lacking functional genes for ICP27 (which is required for growth in all cell types) and ICP34.5 (which is required for growth in nondividing cell types) can deliver a marker gene to both the rodent and primate CNS with high efficiency whilst producing relatively minimal damage and having no effect on sodium currents in dorsal root ganglion neurons. Such viruses paradoxically deliver genes at much higher efficiency than the less disabled single mutant lacking ICP34.5 alone and also, as expected, produce less damage in vivo. Moreover, unlike the single mutant lacking ICP27 the double mutant viruses cannot revert to wild-type by acquistion of complimenting gene sequences during growth of virus stocks in vitro on dividing cells expressing ICP27 since artificial expression of ICP34.5 in these cells is not required. Such ICP27-; ICP34.5- viruses thus offer a platform for the development of vectors which are sufficiently safe for ultimate use in human gene therapy.

Animals↗
PubMed2002

Replication and packaging of helper-dependent adenoviral vectors.

A helper-dependent adenovirus vector (HD AdV) that lacks all the coding sequences of viral proteins from the vector backbone was developed to overcome the problem of host cellular immune responses against E1-deleted AdV. One of the limitations of the current HD AdV system is its relatively inefficient propagation compared with that of E1-deleted AdV, which deters application of the HD AdV, especially in large animal models. We hypothesized that the low titers might be due in part to defects in replication and packaging of the vector DNA. We propagated two HD AdVs with similar marker gene cassettes and stuffer sequences, using two different helper viruses, and determined the replication and packaging efficiencies of viral DNA. Our analysis indicated a difference in replication and packaging efficiencies between the two vectors, which resulted in different propagation efficiencies. Furthermore, dl309, which is similar to the wild-type virus, demonstrated superior helper function over that of the loxP-containing helper virus, AdLC8cluc. These findings may have significant implications for the design of improved production systems of HD AdVs.

Adenoviridae↗
PubMedDate not supplied

Introduction of foreign DNA into mycobacteria using a shuttle phasmid.

Mycobacteria are major pathogens of man and animals. There are approximately 10 million cases of tuberculosis world wide with an annual mortality of three million people. Leprosy, caused by Mycobacterium leprae, afflicts over ten million people, primarily in developing countries. M. tuberculosis and mycobacteria of the M. avium-intracellulare-scrofulaceum (MAIS) group are major opportunistic pathogens of patients with acquired immune deficiency syndrome (AIDS). M. paratuberculosis is the cause of Jöhne's disease in cattle. Yet, BCG (Bacille Calmette-Guerin), an avirulent strain of M. bovis, is the most widely used human vaccine in the world, having been administered to about 2.5 X 10(9) people since 1948 (ref. 4). BCG was highly protective against tuberculosis in England, but has been found not to be effective in preventing pulmonary tuberculosis in adults in Southern India. We have initiated studies to develop the methodology for efficient gene transfer in mycobacteria. We have constructed recombinant shuttle phasmids which are chimaeras containing mycobacteriophage DNA into which an E. coli cosmid is inserted. They can replicate in E. coli as plasmids and in mycobacteria as phages, and transfer DNA across both genera. These shuttle vectors permit for the first time the introduction of foreign DNA by infection into M. smegmatis and BCG. By introducing and ultimately expressing genes for protective antigens for a variety of pathogens, it may be possible to develop cultivatable mycobacteria into useful multivaccine vehicles.

Bacteriophages↗
PubMed2002

[Construction of the mutants of rice nonspecific lipid transfer protein and expression comparison in two kinds of thioredoxin fusion expression vectors].

Five structural important residues of rice nonspecific lipid transfer protein LTP110 were mutated by site-directed mutagenesis. Sequence results showed that they were all mutated successfully. After trying various E. coli expression systems, thioredoxin fusion expression system was found to be a proper system to express wild type and mutant LTP110. cDNA sequences encoding wild type LTP110 and the mutants Y17A, P72L, R46A, D43A, C50A were cloned into two kinds of thioredoxin fusion expression vectors. The expression results were compared. In pTrxFus/GI724 expression system, wild type LTP110 and the mutants Y17A, P72L, R46A could be expressed at low level while D43A and C50A could not be expressed normally; in pET32a(+)/BL21 (DE3) trxB- expression system, wild type LTP110 and all mutant proteins could be expressed very well and the levels were higher than that in pTrxFus/GI724 system. LTP110 fusion protein expressed in pET32a(+) vector was purified and its activity was checked by fluorescence labeled fatty acid. Results indicated that the recombinant LTP110 fusion protein has lipid binding activity. This work provides good basis for the further study.

Amino Acid Sequence↗
PubMed1986

Construction of high, intermediate and low-copy-number promoter-probe plasmids for Bacillus subtilis.

We constructed promoter-probe plasmids for Bacillus subtilis based on the promoterless gene for penicillinase (penP) of Bacillus licheniformis. A Bacillus stearothermophilus DNA fragment, which contained translational stop codons for all three reading frames and transcription terminators of the alpha-amylase gene, was inserted upstream from the Shine-Dalgarno sequence for the penP gene. A low-copy-number plasmid (about 10 copies per chromosome), pPF101, carried tetracycline-resistance gene, whereas another high-copy-number plasmid (about 55 copies per chromosome), pPF201, conferred resistance to kanamycin. The intermediate-copy-number plasmids (about 20 copies per chromosome), pPF001, pPF002, pPF011 and pPF012, conferred chloramphenicol resistance to both B. subtilis and Escherichia coli. Vector plasmids, pPF011 and pPF012, were provided with single cloning sites for BamHI, SacI, SmaI and XbaI upstream from the protein coding region of penP, whereas other plasmids contained only unique BamHI cloning sites. The applicability of these plasmids was demonstrated by cloning a promoter for the alpha-amylase gene.

Bacillus subtilis↗
PubMed2003

The mobile element IS1207 of Brevibacterium lactofermentum ATCC21086: isolation and use in the construction of Tn5531, a versatile transposon for insertional mutagenesis of Corynebacterium glutamicum.

IS1207 is the insertion most frequently found among the spontaneous mutations that abolish the activity of an Escherichia coli phage lambda cI gene integrated in the Corynebacterium Brevibacterium lactofermentum ATCC21086 genome. We examined the transposition of transposon-like structures composed of a selective kanamycin resistance gene (aph3), and one or two IS1207 sequences. One of these, the Tn5531 transposon, transposed efficiently in Corynebacterium glutamicum. A replicative and a non-replicative Tn5531 delivery vector were used in Tn5531 mutagenesis. As IS1207, transposon Tn5531 shows a high frequency of transposition and mutagenesis, and a low target specificity. These features make of Tn5531 an adequate choice for gene identification and gene tagging experiments.

Brevibacterium↗
PubMed2002

Expression and function of the ST2 gene in a murine model of allergic airway inflammation.

BACKGROUND: We have recently reported that soluble ST2 protein levels are elevated in the sera of patients with asthma, and correlate well with the severity of asthma exacerbation. However, the role, function, and kinetics of soluble ST2 expression in asthma remain unclear. OBJECTIVE: The objective of the present study was to clarify the function and kinetics of soluble murine (m) ST2 expression in a murine asthma model. METHODS: We analyzed the kinetics of gene and protein expression of mST2 in sera or lung tissue after allergen (ovalbumin; OVA) challenge in a murine model of allergic airway inflammation, the effects of mST2 protein on OVA-induced Th2 cytokine production in vitro from splenocytes of sensitized mice, and the effects of soluble mST2 on Th2-dependent allergic airway inflammation by in vivo gene transfer of mST2. RESULTS: Serum mST2 protein levels increased to the maximal level 3 h after the allergen challenge, before serum IL-5 levels peaked. The mRNA expression of mST2 in lung tissue was induced after the allergen challenge, while that in the spleen was constitutively detected. Furthermore, pre-treatment with mST2 protein significantly inhibited the production of IL-4 and IL-5, but not IFN-gamma, from OVA-stimulated splenocytes in vitro, and intravenous mST2 gene transfer resulted in a drastic reduction in the number of eosinophils and in the levels of IL-4 and IL-5 in bronchoalveolar lavage fluid, compared with those in response to transfer of non-coding plasmid vector or of lipid alone. CONCLUSION: These results suggest that increases in endogenous mST2 protein after allergen exposure may modulate Th2-mediated airway inflammation, and that in vivo gene transfer of mST2 can be applicable to use in a novel immunotherapy for allergic diseases.

Allergens↗
PubMed1992

Inducible vectors for expression in mammalian cells.

The most recent developments in mammalian cell inducible expression systems have involved the use of bacterial gene control elements and viral transactivator proteins. The combination of hybrid viral transactivator and bacterial repressor proteins, and simple chemical inducers can provide induction ratios of over 1000-fold. These developments will have applications in both cell-based research and the generation of transgenic animals.

Animals↗
PubMed1988

Local administration of cells containing an inserted IL-2 gene and producing IL-2 inhibits growth of human tumours in nu/nu mice.

We have prepared a retroviral expression construct, pPS-IL-2, in which human IL-2 cDNA has been inserted into the polylinker region, and have used the retroviral vector to introduce the functional IL-2 gene into a fibroblast cell line, RAT-1. Peritumoral administration of IL-2-producing RAT-1 cells into congenitally athymic (nu/nu) mice carrying subcutaneous transplants of human carcinoma cells inhibited the growth of the human tumour xenografts.

Animals↗
PubMed1997

Production of bioactive salmon calcitonin from the nonendocrine cell lines COS-7 and CHO.

To produce bioactive salmon calcitonin from the conventional nonendocrine cell lines, COS-7 and CHO, we devised a salmon calcitonin expression vector by combining the amino-terminus of human calcitonin precursor with a salmon calcitonin sequence, inserting the efficient furin-cleavable processing sequence Arg-X-Arg-X-Lys-Arg before salmon calcitonin, and deleting the carboxyl-terminal extension peptide. This chimeric calcitonin precursor terminates at glycine to easily receive an amidation reaction. COS-7 and CHO produced a high level of bioactive calcitonin by the resorption pit formation assay. Although amidating activity is highly expressed in CHO, but only a little in COS-7 cells, both cells produced a similar level of bioactive calcitonin. Thus, the engineered salmon calcitonin expression vector enables nonendocrine cells even with low amidation activity to produce bioactive calcitonin.

Animals↗
PubMed1996

Construction of a stable expression vector carrying sop genes [ZJ1].

Mini-F, the fifth fragment of F plasmid from EcoRI digestion, is known to carry an efficient partitioning function. Two pBR322 plasmid derivatives, pDMC32 and pDMC311, have been constructed from this fragment. The plasmid pDMC32 carries all the relative genes for plasmid stability, ccd, repD, and sop genes (sopA, sopB, and sopC), along with oriS and oriV, while pDMC311 carries only sop genes (sopA, sopB, and sopC). The plasmid maintenance proportions for pDMC32 and pDMC311 in E. coli were 93% and 100%, respectively, after 100 generations continuous cultivation of cells harboring the derivatives, MI32 (pDMC32) and MI311 (pDMC311), in a phosphate-limited basal medium. As a control, the maintenance proportion of plasmid pBR322 dropped down to a low of 10% at generation 55 of continuous cultivation of E. coli MIR322 (pBR322) in the same medium. In order to make a stable expression vector that carries only sop genes, plasmid pDMC40 was constructed by adding a trp promoter from pDR720 to pBR322. The stable expression vector pDMC48 was then derived from pDMC40 by inserting sop genes into it from pDMC311. The maintenance proportion of plasmid pDMC48 in E. coli was still 100% after 100 generations of continuous cultivation of cells harboring the plasmid in phosphate-limited basal medium.

Ampicillin↗
PubMed2002

Adenovirus-based libraries: efficient generation of recombinant adenoviruses by positive selection with the adenovirus protease.

Adenoviruses (Ad) deleted in the protease (PS) gene are capable of only one round of replication in non-complementing cells. This feature was exploited to develop a positive selection method for constructing adenoviral recombinants using ectopic expression of the PS gene in the E1 region. Very low levels of PS were sufficient to ensure the rescue of a PS-deleted Ad genome (Ad(Delta)PS), thereby eliminating deleterious effects PS over-expression might exert on cell or virus growth. In addition to the standard co-transfection method, an alternative protocol was developed in which the Ad5-(Delta)PS viral DNA was delivered by infection before subsequent transfection of 293 cells with the transfer vector. Under optimal conditions, at least one recombinant Ad per 10(3) cells was generated with 100% of the plaques being recombinant. Since the infection/transfection protocol is readily scalable, this represents the first method that allows for the easy construction of adenovirus vector (AdV) libraries with high diversities. This approach addresses in a novel way the bottleneck encountered when converting plasmid libraries, constructed in E. coli using a variety of well-established strategies, into corresponding AdV libraries. It maintains high diversity while generating recombinant viruses with 100% efficiency.

Adenoviridae↗
PubMed1999

Single-step conversion of cells to retrovirus vector producers with herpes simplex virus-Epstein-Barr virus hybrid amplicons.

We report here on the development and characterization of a novel herpes simplex virus type 1 (HSV-1) amplicon-based vector system which takes advantage of the host range and retention properties of HSV-Epstein-Barr virus (EBV) hybrid amplicons to efficiently convert cells to retrovirus vector producer cells after single-step transduction. The retrovirus genes gag-pol and env (GPE) and retroviral vector sequences were modified to minimize sequence overlap and cloned into an HSV-EBV hybrid amplicon. Retrovirus expression cassettes were used to generate the HSV-EBV-retrovirus hybrid vectors, HERE and HERA, which code for the ecotropic and the amphotropic envelopes, respectively. Retrovirus vector sequences encoding lacZ were cloned downstream from the GPE expression unit. Transfection of 293T/17 cells with amplicon plasmids yielded retrovirus titers between 10(6) and 10(7) transducing units/ml, while infection of the same cells with amplicon vectors generated maximum titers 1 order of magnitude lower. Retrovirus titers were dependent on the extent of transduction by amplicon vectors for the same cell line, but different cell lines displayed varying capacities to produce retrovirus vectors even at the same transduction efficiencies. Infection of human and dog primary gliomas with this system resulted in the production of retrovirus vectors for more than 1 week and the long-term retention and increase in transgene activity over time in these cell populations. Although the efficiency of this system still has to be determined in vivo, many applications are foreseeable for this approach to gene delivery.

3T3 Cells↗
PubMed2002

[Construction, packaging and titration of recombinant adeno-associated virus vectors contaning antitumor genes].

OBJECTIVE: To construct, package, and titrate vectors that express antitumor genes using adeno-associated virus (AAV) containing human alpha-fetoprotein (AFP) promoter. METHODS: Recombinant AAV vectors were constructed by blunted ligation of plasmids rAAV-AFP-GFP and pTR-UF5 and p16 cDNA and p21 cDNA. Packaging cell lines, 293 and C12 cells, were transfected with rAAV vectors, plasmid PspRC72, and adenovirus cosmid pAdc with the help of LipofectAMINE and then infected by wild adenovirus 48 hours later. The total viral particle titre was measured by PCR. Electron microscopy was used to observe the morphology of rAAV. C12 cells were infected with rAAV and wild adenovirus (MOI 10) and the titre of infectious virus was measured. by RCA method. Hepatocellular carcinoma cells were cultured and infected with rAAV containing green fluorescent protein (GFP), the infectious rate was observed 48 hours later. RESULTS: Recombinant vectors, rAAV-AFP-p16, rAAV-AFP-p21, rAAV-CMV-p16, and rAAV-CMV-p21, were constructed and verified by DNA sequencing and enzyme digestion. The total viral titre was 10(13) to 10(14)/ml. The titre of infectious rAAV containing GFP was 5 x 10(10)/ml. Electron microscopy showed that the rAVV was round and plaqued with a diameter between 20 and 30 nm. When the MOI was 100, the infectious rate of hepatocellular carcinoma cells was at least 70%. Four rAAV vectors carrying p16 or p21 genes have been constructed. rAAV packaging and titration methods were developed to produce highly-purified AAV stocks. These approaches help in research of gene therapy in liver cancer.

Cell Line↗