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At least 181 records · Page 10Linked to original sources

PubMed1993

An improved retroviral vector for assaying promoter activity. Analysis of promoter interference in pIP211 vector.

We recently developed a novel promoter assay system using a retroviral vector (pIP200 series). Transcription from the internal promoter, which had been inserted for the promoter assay, was shown to be interfered with by transcription from the upstream long terminal repeat (LTR). Here we report a new high-titer 'self-inactivating' vector, in which transcription interference was virtually eliminated. This new vector was constructed by introducing only a very minor mutation into the 'TATA box' in the 3'-LTR. This mutation was successfully transferred to the 5'-LTR after reverse transcription, yielding a provirus incapable of transcribing viral RNA. The viral titer was not reduced by the mutation, permitting general application of this virus.

3T3 Cells↗

PubMed1994

Transfer of the nerve growth factor gene into cell lines and cultured neurons using a defective herpes simplex virus vector. Transfer of the NGF gene into cells by a HSV-1 vector.

Nerve growth factor (NGF) can be expressed in cells by gene transfer using a defective Herpes Simplex virus type 1 (HSV-1) vector. In this report, the defective HSV-1 vector, pHSVngf, is used to infect established cell lines and cultured neurons. Infection of cell lines with pHSVngf results in gene transcription, correct RNA processing, and production of biologically active NGF. Infection of the PC12 neuronal cell line results in the production of biologically active NGF and infection of NGF-dependent neonatal sympathetic neurons in primary culture with pHSVngf leads to neuronal survival in the absence of exogenously-added NGF. NGF expressed by pHSVngf-infected cells does not appear to work through an autocrine intracellular pathway since NGF antibody added to culture media of infected cells could block NGF action. Infection with pHSVngf of cholinergic striatal or septal neurons in dissociated cell culture resulted in an increase in choline acetyltransferase activity. These studies demonstrate the efficacy of defective HSV-1 vectors for delivery and expression of neurotrophin genes in cultured neural cells.

3T3 Cells↗

PubMed1981

Specific-purpose plasmid cloning vectors. I. Low copy number, temperature-sensitive, mobilization-defective pSC101-derived containment vectors.

Two cloning vector plasmids, pHSG415 (7100 bp) and a lambda phage cos site-containing derivative (cosmid) thereof, pHSG422 (8760 bp), were constructed from a low copy number plasmid (pSC101) replicon to permit the propagation of cloned DNA segments at low gene dosage levels. Two features of the vectors, namely temperature sensitivity of replication and inability to be mobilized by conjugative plasmids, cause them to exhibit a high level of "biological containment". The essential characteristics of pHSG415 and pHSG422 may be summarized as follows: (1) their genome copy number is low (4--6 copies/chromosome); (2) their replication ceases at high temperature and they are rapidly lost from host cells grown at temperatures of 37 degrees C and above; (3) the relaxation nick site of pSC101, which is thought to be synonymous with its origin of transfer replication, is absent from the vectors; as a consequence, they are not mobilized to a significant extent by co-existing conjugative plasmids that are able to mobilize wild-type pSC101; (4) they contain unique insertion sites for DNA fragments generated by the following restriction endonucleases: EcoRI, XhoI, XmaI, HindIII and PstI; pHSG415 additionally contains single BamHI, BstEII and HincII sites and may also be used to clone PvuI-generated fragments; (5) the plasmids confer upon their host cells resistance to chloramphenicol, kanamycin and ampicillin, and every unique cloning site, except those of BamHI and BstEII, is located within one of these antibiotic-resistance genes.

Anti-Bacterial Agents↗

PubMed1985

Vectors with restriction-site banks. III. Escherichia coli-Saccharomyces cerevisiae shuttle vectors.

The bank of unique restriction sites present in plasmid pJRD158 has been incorporated into new vectors carrying selective markers and replicons derived from commonly used Escherichia coli-Saccharomyces cerevisiae shuttle vectors pJDB207 and YRp7. The new vectors pMH158 and pJO158 have 21 and 23 unique restriction sites, respectively, and their complete DNA sequences are known.

Base Sequence↗

PubMed1985

Construction of novel shuttle vectors and a cosmid vector for the glutamic acid-producing bacteria Brevibacterium lactofermentum and Corynebacterium glutamicum.

Novel cloning vectors for glutamic acid-producing bacteria have been constructed. Two cryptic plasmids, pAM330 from Brevibacterium lactofermentum and pHM1519 from Corynebacterium glutamicum, were used as precursors, and recombined with pBR325 or pUB110. Resultant composite plasmids were able to propagate and to express the CmR or KmR phenotype in B. lactofermentum and C. glutamicum. A smaller, high-copy-number plasmid, pAJ43, was also isolated following deletion of a part of the pAM330-pBR325 composite plasmid. Furthermore, a cosmid vector, which can be packaged and transduced through phage infection, has been developed using a cohesive-end fragment of the f1A phage and plasmid pAJ43. These plasmids are suitable for use as cloning vectors in the glutamic acid-producing bacteria.

Brevibacterium↗

PubMed1994

The complete nucleotide sequences of the SacBII Kan domain of the P1 pAD10-SacBII cloning vector and three cosmid cloning vectors: pTCF, svPHEP, and LAWRIST16.

The complete nucleotide sequence of the 16,009-bp SacBII Kan domain of the P1 pAD10-SacBII cloning vector and the sequences of three cosmid cloning vectors, pTCF (7941 bp), svPHEP (9201 bp), and LAWRIST16 (5194 bp) have been determined. A modified diatomaceous earth (Prep-A-Gene)-based procedure, which rapidly yields highly supercoiled double-stranded DNA from recombinant P1 and cosmid clones suitable for generating shotgun libraries, also has been developed. The isolated recombinant DNAs were physically sheared to generate 1- to 2-kb fragments that then were blunt-ended and subcloned into double-stranded pUC-based sequencing vectors. The double-stranded sequencing templates were isolated by an alkaline lysis method and subjected to Taq polymerase catalyzed fluorescent end-labeled primer cycle sequencing. After shotgun sequence assembly, contig gaps were closed and ambiguities were resolved via Sequenase catalyzed fluorescent dye-terminator sequencing.

Bacteriophage P1↗

PubMed2004

HSV vector-delivery of GDNF in a rat model of PD: partial efficacy obscured by vector toxicity.

Herpes simplex virus (HSV)-derived vectors have been suggested for potential use in gene therapy for Parkinson's disease (PD). HSV naturally infects adult neuronal cells and possesses a large genome for the insertion of transgenes. In the present study, we have used two different HSV constructs to deliver glial cell line-derived neurotrophic factor (GDNF) to the striatum, and to assess the neuroprotective effects of the GDNF product in an intrastriatal 6-hydroxydopamine lesion model. One construct is blocked for IE gene expression whereas the other is deleted in the thymidine kinase gene. Both constructs induced a significant protection of the dopaminergic neurons in the substantia nigra from the lesions, whereas only one induced a transient behavioural recovery in amphetamine-induced rotation. Unexpectedly, the more deleted virus caused the greater toxicity. This was found to be due to the way the vector was purified. The issue of toxicity, which might account for the variable functional effects, needs resolving prior to therapeutic application of these vectors.

Animals↗

PubMed2003

Adeno-associated viral vector gene expression in the adult rat spinal cord following remote vector delivery.

The current investigation tests whether adeno-associated viral vectors (rAAV) undergo remote delivery to the spinal cord via peripheral nerve injection as previously demonstrated with adenoviral vectors. The sciatic nerves of adult rats (n = 10) were injected with either an rAAV (rAAVCMV-lacZ) or adenoviral (AdCMV-lacZ) vector (1.4 x 10(7) particles/ml). After 21 days, the rAAV group demonstrated significantly higher spinal cord viral expression than the adenoviral group (P < 0.024). A second group of rats was injected with rAAV expressing the green fluorescence protein (GFP) reporter gene. GFP was detected 21 days after unilateral sciatic nerve injection in the neurons of the dorsal root ganglion and spinal cord. The codistribution of the viral genome and transgene in CNS neurons was confirmed with in situ hybridization. In summary, rAAV genes are expressed in CNS neurons following peripheral nerve injection at levels exceeding those seen following remote adenovirus injection.

Animals↗

PubMed2000

Protective copolymers for nonviral gene vectors: synthesis, vector characterization and application in gene delivery.

Uncontrolled interactions of gene vectors and drug carriers in and with an in vivo environment pose serious limitations to their applicability. In order to reduce such interactions we have designed, synthesized and applied novel copolymers of poly(ethylene glycol) and reactive linkers which are derivatized with anionic peptides after copolymerization. The anionic copolymer derivatives are used to coat positively charged nonviral gene vectors by electrostatic interactions. The copolymer coat confers to polyelectrolyte colloids of DNA and polycations steric stabilization in their minimal size and prevents salt- and serum albumin-induced aggregation. Furthermore, complement activation and the interaction with serum proteins are drastically reduced or abolished in contrast to unprotected DNA complexes. The designed vectors are compatible with the intracellular steps of gene delivery and can even enhance transfection efficiency as demonstrated with various adherent and nonadherent cell lines in culture. The synthetic concept is amenable to the principles of combinatorial chemistry and the copolymeric products may be applicable beyond gene delivery in targeted drug delivery. Gene Therapy (2000) 7, 1183-1192.

Cells, Cultured↗

PubMed2005

Retroviral vector backbone immunogenicity: identification of cytotoxic T-cell epitopes in retroviral vector-packaging sequences.

Retroviral vectors are the frequently applied gene delivery vehicles for clinical gene therapy, but specificity of the immunogenicity to the protein encoded by the inserted gene of interest is a problem which needs to be overcome. Here, we describe human cytotoxic T-lymphocyte (CTL) clones recognizing epitopes derived from the protein encoded by the retroviral vector backbone, which were established during the course of our attempts to generate CTLs against cytomegalovirus (CMV) or human papilloma virus (HPV) in vitro. In the case of healthy CMV-seronegative donors, CTL lines specific for retrovirally transduced cells were generated in four out of eight donors by stimulating CD8 T cells with CD40-activated B (CD40-B) cells retrovirally transduced with CMV-pp65. Two CTL clones derived from one of the CTL lines were found to recognize epitopes from gag in the context of HLA-B(*)4403 and -B(*)4601, respectively. Similarly, an HLA-B(*)3501-restricted CTL clone from a cervical cancer patient recognized an epitope located in the junctional regions of the gag and pol sequences. These results show that polypeptides encoded by components of the retroviral vector backbone are in fact immunogenic, generating CTLs in vitro in human cells. Thus, potential CTL responses to retroviral products should also be considered in clinical settings.

Animals↗

PubMed1990

Biotechnology as applied to vectors and vector control.

Vector borne diseases remain some of the major public health problems in the developing world. In this review some of the current applications of biotechnology to vectors and vector control and some of the possible future applications are discussed.

Animals↗

PubMed2001

Transduction of ovarian cancer cells: a recombinant adeno-associated viral vector compared to an adenoviral vector.

Recombinant adeno-associated virus (rAAV) vectors have emerged as vehicles for gene therapy. In addition, anti-neoplastic properties have been attributed to wild-type AAV. To take advantage of both features and to overcome technical problems associated with rAAV preparation, we developed a production method in which rAAV particles are amplified in an infectious cycle in the presence of wtAAV. This results in a 10(3)-10(4)-fold amplification of rAAV input particles. rAAV-GFP particles generated by this method were used to transduce ovarian cancer cell lines to evaluate their potential in ovarian cancer gene therapy, in comparison to a rAd-GFP vector. The transduction efficiency of NIH-OVCAR3, MDAH 2774 and SKOV3 cells with rAAV-GFP particles was low (< 1%) and did not improve by increasing the number of particles/cell. Repeated administration and continued exposure of NIH-OVCAR3 and MDAH 2774 improved transduction to over 3%. In contrast, these cell lines were more efficiently transduced by rAAV-GFP in the presence of adenovirus (approximately 15%) and by rAd-GFP (> 50%). These results indicate that in contrast to rAd vectors, rAAV particles are not suitable for therapeutic gene transfer in ovarian cancer cells unless efficient help can be provided to mediate ss to ds DNA conversion.

Adenoviridae↗

PubMed1984

A Bacillus subtilis secretion vector system derived from the B. subtilis alpha-amylase promoter and signal sequence region, and secretion of Escherichia coli beta-lactamase by the vector system.

A secretion vector system in Bacillus subtilis was constructed from the alpha-amylase promoter and signal sequence coding region of an alpha-amylase hyperproducing strain, B. subtilis NA64, and the major part of the plasmid pTUB4 which was derived from pUB110. When an Escherichia coli beta-lactamase gene, lacking its own promoter and signal sequence coding region, was introduced into the secretion vector system, beta-lactamase was expressed in B. subtilis. In addition, more than 95% of the enzyme synthesized was secreted into the culture medium via the secretion vector system. Secreted beta-lactamase crossreacted with rabbit antiserum raised against the E. coli enzyme.

Bacillus subtilis↗

PubMed2000

Tissue-binding properties of a synthetic peptide DNA vector targeted to cell membrane integrins: a possible universal nonviral vector for organ and tissue transplantation.

BACKGROUND: Gene delivery through a nonviral, receptor-mediated system widely expressed in transplanted tissue would have important advantages in transplantation, where gene delivery is performed ex vivo. Integrins are widely expressed cell surface receptors and can be targeted for gene delivery. METHODS: A synthetic 31 amino acid DNA vector (polylysine-molossin) comprising a 15-amino acid moiety for targeting cellular integrins (derived from the snake venom, molossin) and a 16-amino acid polylysine moiety for DNA-binding, has been evaluated. The 31-amino acid vector, as well as its separate 15-amino acid integrin-binding and (lys)16 components, were individually synthesized, and a monoclonal antibody was raised to the molossin peptide for these studies. Binding to cell lines and tissue sections and capacity for gene delivery were examined. RESULTS: Flow cytometric studies with the ECV304 cell line demonstrated that the binding of polylysine-molossin and polylysine-molossin/DNA complexes involved both electrostatic and integrin-mediated interactions with the cells, with the electrostatic binding being sufficient for maximal binding. However, binding to cellular integrins was essential for successful gene transfer. Binding studies on frozen tissue sections of the rat and pig demonstrated that the molossin peptide bound to many cell types of interest in transplantation, but not to all. Among the negative tissues were vascular endothelium and pancreatic islets. Small species differences in tissue binding were noted between the rat and pig. CONCLUSIONS: This study defines the cooperative nature of the binding of this vector system to target cells and establishes the cell types most likely to be effectively targeted for DNA transfer.

Antineoplastic Agents↗

PubMed2004

The PRESAT-vector: asymmetric T-vector for high-throughput screening of soluble protein domains for structural proteomics.

A rapid unidirectional method for cloning PCR-amplified cDNA fragments into virtually any fusion protein expression vector is described. The method, termed PRESAT-vector cloning, is based on a T-vector technique that does not require restriction endonuclease digestion of the PCR product. Subsequently, we applied a novel ORF selection method of the ligated plasmid products. This second step involves restriction endonuclease treatment that eliminates the plasmids containing an ORF in the wrong orientation prior to transformation into the bacterial host for further protein expression studies. To achieve this selection, we customized the 5'-sequence of the "rear" PCR primer corresponding to the C terminus of the protein to be expressed. The colonies harbored only the ligated products of the desired orientation at >90% efficiency. This method is applied to a GST fusion expression system, and an HTS system for soluble proteins from an expression library was tested.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2006

Review of the Minimus Complex of Anopheles, main malaria vector in Southeast Asia: from taxonomic issues to vector control strategies.

BACKGROUND: The Minimus Complex of Anopheles subgenus Cellia is composed of two sibling species, A and C, on the Southeast Asian mainland, and a third allopatric species E that occurs in the Ryukyu Archipelago (Japan), a malaria-free region. Anopheles minimus s.l. is considered to be one of the main malaria vector in the hilly forested regions of Southeast Asia. Despite a large number of studies over its range of distribution, it is difficult to have a global view of the ecological and bionomical characteristics of the individual species as different identification methods were used, generally without specific identification of the sibling species. OBJECTIVES: (1) To review the main malaria studies on An. minimus s.l.; (2) to discuss recently published data on the biology and ecology of each sibling species; and (3) to identify gaps in our understanding of the Minimus Complex. REVIEW RESULTS: Major biological and ecological trends are addressed, such as the high plasticity of trophic behaviour and the sympatry of species A and C over the Southeast Asian mainland. Despite the availability of rapid molecular identification methods, we still lack important information concerning the biological characteristics of each sibling species. These gaps must be filled in the future because An. minimus species A and C may exhibit different abilities to transmit malaria. CONCLUSION: We expect that entomological surveys will employ molecular methods to clearly identify these two species, and thus elucidate the biological characteristics of each species. As a consequence, current vector control strategies will be improved by targeting the most efficient vectors.

Animals↗

PubMed1997

Psi- vectors: murine leukemia virus-based self-inactivating and self-activating retroviral vectors.

We have developed murine leukemia virus (MLV)-based self-inactivating and self-activating vectors to show that the previously demonstrated high-frequency direct repeat deletions are not unique to spleen necrosis virus (SNV) or the neomycin drug resistance gene. Retroviral vectors pKD-HTTK and pKD-HTpTK containing direct repeats composed of segments of the herpes simplex virus type 1 thymidine kinase (HTK) gene were constructed; in pKD-HTpTK, the direct repeat flanked the MLV packaging signal. The generation of hypoxanthine-aminopterin-thymidine-resistant colonies after one cycle of retroviral replication demonstrated functional reconstitution of the HTK gene. Quantitative Southern analysis indicated that direct repeat deletions occurred in 57 and 91% of the KD-HTTK and KD-HTpTK proviruses, respectively. These results demonstrate that (i) deletion of direct repeats occurs at similar high frequencies in SNV and MLV vectors, (ii) MLV psi can be efficiently deleted by using direct repeats, (iii) suicide genes can be functionally reconstituted during reverse transcription, and (iv) the psi region may be a hot spot for reverse transcriptase template switching events.

Blotting, Southern↗

PubMed2004

Cross-packaging of human immunodeficiency virus type 1 vector RNA by spleen necrosis virus proteins: construction of a new generation of spleen necrosis virus-derived retroviral vectors.

The ability of the nonlentiviral retrovirus spleen necrosis virus (SNV) to cross-package the genomic RNA of the distantly related human immunodeficiency virus type 1 (HIV-1) and vice versa was analyzed. Such a model may allow us to further study HIV-1 replication and pathogenesis, as well as to develop safe gene therapy vectors. Our results suggest that SNV can cross-package HIV-1 genomic RNA but with lower efficiency than HIV-1 proteins. However, HIV-1-specific proteins were unable to cross-package SNV RNA. We also constructed SNV-based gag-pol chimeric variants by replacing the SNV integrase with the HIV-1 integrase, based on multiple sequence alignments and domain analyses. These analyses revealed that there are conserved domains in all retroviral integrase open reading frames (orf), despite the divergence in the primary sequences. The transcomplementation assays suggested that SNV proteins recognized one of the chimeric variants. This demonstrated that HIV-1 integrase is functional in the SNV gag-pol orf with a lower transduction efficiency, utilizing homologous (SNV) RNA, as well as the heterologous vector RNA of HIV-1. These findings suggest that homology in the conserved sequences of the integrase protein may not be fully competent in the replacement of protein(s) from one retrovirus to another, and there are likely several other factors involved in each of the steps related to replication, integration, and infection. However, further studies to dissect the gag-pol region will be critical for understanding the mechanisms involved in the cleavage of reverse transcriptase, RNase H, and integrase. These studies should provide further insight into the design and development of novel molecular approaches to block HIV-1 replication and to construct a new generation of SNV-based vectors.

Amino Acid Sequence↗