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At least 415 records · Page 23Linked to original sources
PubMed2002

Modification of multiple transcriptional regulatory elements in a Moloney murine leukemia virus gene transfer vector circumvents silencing in fibroblast grafts and increases levels of expression of the transferred enzyme.

Down-regulation of retroviral vector expression occurs in a number of cell types after transplantation. Although a number of vector elements have been shown to affect expression in specific experimental situations, the results can vary depending on the specific cDNA being expressed, the individual retroviral elements included in vectors, the promoter, or the inclusion of selectable markers. In previous experiments with the lysosomal enzyme beta-glucuronidase, silencing has occurred in more than 95% of transduced cells regardless of the position of the expression unit within the vector, whether a eukaryotic or viral promoter was used, whether a bacterial selectable marker gene was present or not, the target cell type, or the species of the host. It has been a consistent finding that a small number of continuously expressing cells persist for long periods after transplantation. In this study we found that deletion of all the transcriptional regulatory elements from the vector LTR, inclusion of a permissive primer binding site sequence, and use of a eukaryotic housekeeping promoter could greatly increase the number of expressing cells in fibroblast grafts in subcutaneous neo-organs and in the brain. Furthermore, the level of enzyme expression was increased five-fold on a per positive cell basis, indicating that the deleted regulatory elements were exerting a negative effect on expression in the few cells that were positive before modification of the vector. This resulted in more than a 50-fold increase in total activity compared with the previous highest expressing vector.

Animals↗
PubMed1986

Efficient introduction of plasmid DNA into human hemopoietic cells by encapsidation in simian virus 40 pseudovirions.

Introduction of DNA into human hemopoietic cells is required for the study of regulatory mechanisms operating in these cells, as well as for possible procedures of gene therapy. However, with hemopoietic cells the conventional technique of calcium phosphate precipitation is inefficient. The pathway of encapsidation of plasmid DNA as simian virus 40 (SV40) pseudovirions for the introduction of new genetic material was therefore investigated. Encapsidation was achieved in COS (monkey kidney) cells, which express SV40 large tumor (T) antigen constitutively. The vector, pSO, was introduced to the COS cells by DNA transfection. It carried the SV40 origin of replication (ori), to facilitate replication of the plasmid in the COS cells. The SV40 capsid proteins were supplied in trans by a helper SV40 virus. The bacterial chloramphenicol acetyltransferase gene cat was used as a model for gene transmission. After encapsidation, the pseudovirions were used in infection of the human erythroleukemic cell line K562 and of normal human bone marrow cells. The results demonstrate that the cat gene can be transmitted with high efficiency. Over 40% of the infected K562 cells and 30% of the infected bone marrow cells were observed to contain plasmid DNA 48 hr after infection. Moreover, the results suggest that the efficiency of gene transmission by this vector can be improved and so may approach the theoretical 100%.

Acetyltransferases↗
PubMed1985

Construction of a vector, pRSVcatamb38, for the rapid and sensitive assay of amber suppression in human and other mammalian cells.

We describe the generation of an amber mutation in the chloramphenicol acetyltransferase (cat) gene of the mammalian cell transfection vector pRSVcat (Gorman et.al. (1982), Proc.Natl.Acad.Sci. 79 6777-6791). We have demonstrated the in vivo suppression of this amber mutation in monkey and human cells by co-transfection with a synthetic Xenopus suppressor tRNATyr under the control of the late SV40 promoter. The vector, pRSVcatamb38, may be used to quantitate amber suppression in various mammalian cells.

Acetyltransferases↗
PubMed2000

Somatic gene therapy for hypertension.

Gene therapy for hypertension is needed for the next generation of antihypertensive drugs. Current drugs, although effective, have poor compliance, are expensive and short-lasting (hours or one day). Gene therapy offers a way to produce long-lasting antihypertensive effects (weeks, months or years). We are currently using two strategies: a) antisense oligodeoxynucleotides (AS-ODN) and b) antisense DNA delivered in viral vectors to inhibit genes associated with vasoconstrictive properties. It is not necessary to know all the genes involved in hypertension, since many years of experience with drugs show which genes need to be controlled. AS-ODN are short, single-stranded DNA that can be injected in naked form or in liposomes. AS-ODN, targeted to angiotensin type 1 receptors (AT1-R), angiotensinogen (AGT), angiotensin converting enzyme, and ss1-adrenergic receptors effectively reduce hypertension in rat models (SHR, 2K-1C) and cold-induced hypertension. A single dose is effective up to one month when delivered with liposomes. No side effects or toxic effects have been detected, and repeated injections can be given. For the vector, adeno-associated virus (AAV) is used with a construct to include a CMV promoter, antisense DNA to AGT or AT1-R and a reporter gene. Results in SHR demonstrate reduction and slowing of development of hypertension, with a single dose administration. Left ventricular hypertrophy is also reduced by AAV-AGT-AS treatment. Double transgenic mice (human renin plus human AGT) with high angiotensin II causing high blood pressure, treated with AAV-AT1-R-AS, show a normalization of blood pressure for over six months with a single injection of vector. We conclude that ODNs will probably be developed first because they can be treated like drugs for the treatment of hypertension with long-term effects. Viral vector delivery needs more engineering to be certain of its safety, but one day may be used for a very prolonged control of blood pressure.

Animals↗
PubMed1997

Modelling immunocontraception in disseminating systems.

Vectored immunocontraception is a novel technology and simple models are described to help predict whether, and how, it might work. That is, given that an effective immunocontraceptive agent can be produced, and given that it can be inserted into a microparasitic or macroparasitic infective vector, would the vector persist and reach a high prevalence in the host and, if so, would it sterilize a sufficient proportion of the host breeding population to significantly reduce its density? Both conditions are necessary for success. The first question is an epidemiological one, relating solely to disseminating systems and differing according to whether the vector itself is newly introduced or pre-existing. If it is newly introduced, the assumption is that it is present in some other geographical areas occupied by the same target species, or is found in closely-related species. If the vector already exists in the population, the issue is one of competition between the engineered and wild-type vectors. The second question is an ecological one, common to both non-disseminating and disseminating systems. Whatever level of sterilization the immunocontraception provides must translate into a significant reduction in population density, having regard to the nature and extent of compensatory, density-dependence mechanisms in the population. These two questions, together with other more minor issues, are addressed in turn with particular reference to models for immunocontraception of brushtail possums (Trichosurus vulpecula) in New Zealand.

Animals↗
PubMed2000

A novel episomal shuttle vector for transformation of Cryptococcus neoformans with the ccdB gene as a positive selection marker in bacteria.

We report the engineering of a new shuttle vector featuring its episomal maintenance in Cryptococcus neoformans and the lethal Escherichia coli ccdB gene for positive selection in bacteria. Telomere-like sequences from C. neoformans and the STAB fragment confer episomal maintenance to the vector (pPM8) upon transformation in C. neoformans. The vector generated high transformation frequencies and each transformant was estimated to harbor thirty copies of the plasmid. The plasmids recovered in E. coli from the C. neoformans transformants showed no evidence of rearrangement. This construct will be very useful for cloning and studying the regulation of genes in C. neoformans.

Bacterial Proteins↗
PubMed2003

Genetic stability of attenuated mengovirus vectors with duplicate primary cleavage sequences.

Short poly(C)-tract Mengoviruses have proven vaccine efficacy in many species of animals. A novel vector for the delivery of foreign proteins was created by insertion of a second autoproteolytic primary cleavage cassette linked to a multiple cloning site (MCS) into an attenuated variant of Mengo. Nineteen cDNAs from foreign sequences that ranged from 39 to 1653 bases were cloned into the MCS. The viral reading frame was maintained and translation resulted in dual, autocatalytic excision of the foreign peptides without disruption of any Mengo proteins. All cDNAs except those with the largest insertions produced viable virus. Active proteins such as GFP, CAT, and SIV p27 were expressed within infected cells. Relative to parental Mengo, the growth kinetics and genetic stability of each vector was inversely proportional to the size of the inserted sequence. While segments up to 1000 bases could be carried, inserts greater than 500-600 bases were usually reduced in size during serial passage. The limit on carrying capacity was probably due to difficulties in virion assembly or particle stability. Yet for inserts less than 500-600 bases, the Mengo vectors provided an effective system for the delivery of foreign epitopes into cells and mice.

Animals↗
PubMed2001

Gene therapy for hypertension: sense and antisense strategies.

Gene therapy for hypertension is needed for the next generation of antihypertensive drugs. Current drugs, although effective, have poor compliance, are expensive and short-lasting (hours or one day). Gene therapy offers a way to produce long-lasting antihypertensive effects (weeks, months or years). We are currently using two strategies: antisense oligodeoxynucleotides (AS-ODN), an dantisense DNA delivered in viral vectors, to inhibit genes associated with vasoconstrictive properties. It is not necessary to know all the genes involved in hypertension, since many years of experience with drugs show which genes need to be controlled. AS-ODNs are short, single-stranded DNA that can be injected in naked form or in liposomes. AS-ODNs, targeted to AT1 receptors (AT1R), angiotensinogen (AGT), angiotensin converting enzyme (ACE) and beta 1-adrenergic receptors effectively reduce hypertension in rat models (SHR, 2K-1C and cold-induced) hypertension. The effects can last up to one month when delivered with liposomes. No side effects or toxic effects have been detected and repeated injections can be given. For the vector, adeno-associated virus (AAV) is used with a construct to include a CMV promoter, antisense DNA to AGT or AT1R and a reporter gene. Results in SHR demonstrate reduction and slowing of hypertension development with a single dose administration. Left ventricular hypertrophy is also reduced by AAV-AS-AGT treatment. Double transgenic mice (human renin plus human AGT) with high angiotensin II (Ang II) causing high blood pressure, treated with AAV-AT1R-AS, show a normalisation of blood pressure for over 6 months with a single injection of vector. We conclude that ODNs will probably be developed first because they can be treated like drugs for the treatment of hypertension with long-term effects. Viral vector delivery needs more engineering to be certain of its safety but one day may be used for a very prolonged control of blood pressure.

Angiotensinogen↗
PubMed1995

A replacement vector used to introduce subtle mutations into mouse genes.

A replacement vector convenient for introducing subtle mutations into various mouse genes has been developed using, a model system, the mouse transthyretin-encoding gene (ttr) and mouse embryonal carcinoma F9 cells. The vector consists of part of ttr carrying a subtle mutation in its second exon, and a cassette of the neomycin-resistance (neo)- and herpes simplex virus thymidine kinase (HSV-tk)-encoding genes flanked with a 3-kb duplication of mostly the second intron of ttr. In the first step ('replacement'), part of the endogenous ttr was replaced by vector DNA via homologous recombination, and two such clones, #33 and #77, were isolated from 185 G418-resistant clones by allele-specific PCR. In the second step ('excision'), gancyclovir-resistant colonies were screened, and 7 and 84% of those isolated from clones #33 and #77, respectively, were demonstrated to carry the subtle mutation in ttr, without the cassette of selection markers. In five independently isolated random integrants of the same vector DNA, the cassette of selection markers was excised efficiently by recombination within the duplication.

Animals↗
PubMed2002

Encapsulation of recombinant adenovirus into alginate microspheres circumvents vector-specific immune response.

Pre-existing immunity against adenoviruses may compromise the efficacy of adenoviral vectors for vaccination and gene therapy. The purpose of this study was to determine whether encapsulation of adenovirus recombinants into biodegradable alginate microparticles could circumvent the vector-specific immune response. Mice were immunized either intranasally (i.n.) or intraperitoneally (i.p.) with human adenovirus type 5 (HAd5), resulting in the development of virus-specific antibodies. Immunized and nai;ve mice were inoculated with AdCA36lacZ (an E1-deleted HAd5 recombinant containing the bacterial beta-galactosidase (LacZ) gene), encapsulated (E) into alginate microparticles, or nonencapsulated (NE) ie, as a virus suspension. LacZ expression in animals immunized once (1x) or twice (2x) with HAd5 and subsequently inoculated with NE-AdCA36lacZ (NE-Z) was significantly (P<0.001) reduced compared to those levels observed in NE-Z inoculated nai;ve mice, suggesting that the immune response against the vector adversely affected transgene expression. In contrast, there was only slight reduction (P>0.05) in LacZ expression in mice immunized 1x or 2x with HAd5 that were subsequently inoculated with E-AdCA36lacZ (E-Z) compared to those levels obtained in E-Z inoculated nai;ve animals. Similar results were obtained with i.n. or i.p. inoculated animals. These results indicate that microencapsulation of recombinant adenovirus effectively circumvented the vector-specific immune response.

Adenoviridae↗
PubMed2003

Construction and characterization of pentacistronic retrovirus vectors.

The picornavirus foot-and-mouth disease virus 2A sequence was combined with three different internal ribosome entry segments to construct and characterize three independent pentacistronic retroviruses of different sizes. Efficient co-expression of the five proteins was successful and titres obtained for these pentacistronic virus vectors (final genome size approximately 7.9 kb) were comparable to those of vector systems with shorter genomes. Other vectors constructed that exceeded the genome length of the wild-type virus suffered frequent deletions.

3T3 Cells↗
PubMed2003

Characterization of a recombinant Newcastle disease virus expressing the green fluorescent protein.

A recombinant Newcastle disease virus (NDV) expressing the green fluorescent protein (GFP) was generated by applying reverse genetics techniques. The GFP open reading frame flanked by NDV transcription start and stop sequences was inserted between the fusion (F)- and hemagglutinin-neuraminidase genes in a full-length cDNA clone of NDV. This plasmid transcribing antigenome RNA was cotransfected with helper plasmids expressing viral nucleoprotein, phosphoprotein and large protein into cells stably expressing T7 RNA polymerase. The rescued virus was first propagated in embryonated eggs and the allantoic fluid was used to infect cells. Northern blot analysis of RNA isolated from infected cells demonstrated the proper transcription of the introduced GFP-mRNA. The appearance of GFP in live infected cells confirmed further the recovery of a recombinant NDV (rNDVGFP1) expressing the reporter gene. The expression of the heterologous gene was maintained stably for at least five passages in embryonated eggs. The replication kinetics in embryonated eggs and pathogenicity in chickens of rNDVGFP1 did not differ significantly from that of the parent virus. Using GFP autofluorescence, virus infected cells could be tracked easily in native preparations, organ explants and primary tracheal cell cultures. Taken together, these data demonstrate the use of GFP-expressing recombinant NDV for analysis of NDV dissemination and pathogenesis and indicate the potential usefulness of NDV as a vaccine vector.

Animals↗
PubMed1995

Targeted mutagenesis in mammalian cells mediated by intracellular triple helix formation.

As an alternative to standard gene transfer techniques for genetic manipulation, we have investigated the use of triple helix-forming oligonucleotides to target mutations to selected genes within mammalian cells. By treating monkey COS cells with oligonucleotides linked to psoralen, we have generated targeted mutations in a simian virus 40 (SV40) vector contained within the cells via intracellular triple helix formation. Oligonucleotide entry into the cells and sequence-specific triplex formation within the SV40 DNA deliver the psoralen to the targeted site. Photoactivation of the psoralen by long-wavelength UV light yields adducts and thereby mutations at that site. We engineered into the SV40 vector novel supF mutation reporter genes containing modified polypurine sites amenable to triplex formation. By comparing the abilities of a series of oligonucleotides to target these new sites, we show that targeted mutagenesis in vivo depends on the strength and specificity of the third-strand binding. Oligonucleotides with weak target site binding affinity or with only partial target site homology were ineffective at inducing mutations in the SV40 vectors within the COS cells. We also show that the targeted mutagenesis is dependent on the oligonucleotide concentration and is influenced by the timing of the oligonucleotide treatment and of the UV irradiation of the cells. Frequencies of intracellular targeted mutagenesis in the range of 1 to 2% were observed, depending upon the conditions of the experiment. DNA sequence analysis revealed that most of the mutations were T.A-to-A.T transversions precisely at the targeted psoralen intercalation site. Several deletions encompassing that site were also seen. The ability to target mutations to selected sites within mammalian cells by using modified triplex-forming oligonucleotides may provide a new research tool and may eventually lead to therapeutic applications.

Animals↗
PubMed1997

Recombinant viral vector vaccines for the veterinary use.

Recently, genetically engineering using recombinant DNA techniques has been applied to design new viral vaccines in order to reduce some problems which present viral vaccines have. Up to now, many viruses have been investigated for development of recombinant attenuated vaccines or live viral vectors for delivery of foreign immunogenic antigens. In this review, we introduced three kind of viruses; herpesviruses, vaccinia viruses, and adenoviruses, which have best widely been studied as recombinant vaccines or delivery vaccines for the veterinary use.

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↗
PubMed1986

Promoter-probe vectors for the analysis of divergently arranged promoters.

A series of plasmid-based promoter-probe vectors has been constructed which are particularly useful for the analysis of divergent control regions. Each vector contains a pair of divergently oriented indicator genes whose expression can be monitored over a wide range by simple assay methods. These genes are separated by different polylinkers. Specifically, the beta-galactosidase gene (lacZ) was employed in combination with either the galactokinase gene (galK) or the alkaline phosphatase gene (phoA). In all cases translational stop codons are present in all three reading frames upstream from the initiation codon. The vectors permit direct detection of promoters--independent of insert orientation--on indicator plates after transformation. Using this vector system, we further characterized the divergent tet control regions of transposon Tn10 and plasmid pBR322.

Alkaline Phosphatase↗