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At least 433 records · Page 24Linked to original sources
PubMed1990

Construction of a series of pSAM2-based integrative vectors for use in actinomycetes.

We have developed vectors which allowed integration of cloned DNA at a single site in the chromosome of Streptomyces lividans 66. These vectors made use of (1) an Escherichia coli replicon, (2) a thiostrepton (Th)- and a streptomycin/spectinomycin-resistance gene for selection in Streptomyces, (3) a 3.5-kb fragment of the Streptomyces integrative plasmid pSAM2 containing its xis and int genes as well as its attachment site, attP, to direct the integration of the vectors at the chromosomal pSAM2 attachment site attB, (4) the origin of transfer of the IncP broad-host-range plasmid RK2 which allowed the mobilization of the vectors from E. coli to S. lividans, and (5) the Th-inducible tipA promoter to permit regulated transcription of cloned genes. We demonstrated that pPM927, a plasmid which contained all of these elements, was able to transfer cloned fragments from E. coli to S. lividans by conjugation, stably integrate into the chromosome, and express cloned genes from the tipA promoter. Furthermore, since pPM927 contained the pBR322 replicon, cloned fragments could be conveniently recovered from the S. lividans chromosome for analysis in E. coli by cleavage of genomic DNA isolated from transformed strains, intramolecular ligation and transformation. Since we have shown that the pSAM2 attB site forms part of a conserved prokaryotic tRNA gene, these integrative vectors are potentially useful tools for analysis and expression of genes in diverse bacteria.

Actinomycetales↗
PubMed2001

Degenerated pIX-IVa2 adenoviral vector sequences lowers reacquisition of the E1 genes during virus amplification in 293 cells.

A critical issue for E1-deleted adenoviral vectors manufactured from 293 cells is the emergence of replication-competent adenovirus (RCA). These contaminants arise through homologous recombination between identical sequences framing the E1 locus displayed by 293 cells, and the vector backbones. Modified recombinogenic sequences (syngen) were thus introduced within the vector backbone, and virus viability and RCA emergence were assessed. Syngen#1 is a synthetic sequence displaying silent point mutations in the pIX and IVa2 coding regions. A side by side comparison of Ad5CMV/p53 (E1-deleted adenovirus expressing the p53 tumor suppressor gene) and AVdeltaE1#1CMV/p53 (with syngen#1 in place of wild-type sequences) demonstrated a normal productivity for the modified construct. The altered sequences did not impair p53-mediated apoptosis in a model tumor cell line. Most importantly, a statistically significant decrease in terms of RCA occurrence could also be demonstrated. Degenerescence of the recombinogenic sequences could be further accentuated by modifying noncoding pIX region (syngen #2), with no effect on virus productivity and stability. We concluded that these vector modifications constitute a feasible strategy to reduce RCA emergence during amplification in 293 cells. This approach could also be applied to decrease reincorporation of the E1 genes during amplification of deltaE1deltaE4 vectors in 293/E4-trans-complementing cells.

Adenoviridae↗
PubMed1985

Phasmid vectors for identification of genes by complementation of Escherichia coli mutants.

A bacteriophage lambda cloning vector was designed to facilitate the isolation of genes from procaryotic organisms by complementation of Escherichia coli mutants. This vector, lambda SE4, was constructed by attaching a very-low-copy-number replication system (from the plasmid NR1) and a spectinomycin resistance gene to the left arm of lambda 1059 (Karn et al., Proc. Natl. Acad. Sci. U.S.A. 77:5172-5176, 1980). This phasmid cloning vector is capable of growing lytically as a phage in a nonimmune host or lysogenically as a phasmid in an immune host. This phasmid utilizes the Spi- selection for insertions of DNA into the vector and has the ability to accept 2- to 19-kilobase Sau3A1, BamHI, BglII, BclI, or XhoII fragments; recombinants lysogenize immune hosts as single-copy-number selectable plasmids at 100% frequency. An E. coli library was constructed by using the initial vector lambda SE4, and clones of a number of representative genes were identified. A typical clone, lambda ant+, was shown to be readily mutagenized by a mini-Tn10 transposon. A general method for transferring cloned DNA segments onto bacteriophage lambda was developed. The method involves the use of in vivo recombination with a selection and was used to construct two derivatives of lambda SE4. Possible uses of these vectors and of the method for transferring cloned DNA onto phage lambda are discussed.

Bacteriophage lambda↗
PubMed1989

Vectors permitting visual monitoring of simple transposition events.

The construction and use of two novel transposon(Tn)-delivery vectors is described. These vectors carry Inc.W or Inc.N broad-host-range transfer functions cloned next to the narrow-host-range replicon of pBR329. The host specificities of pSLX10 and pSLX23 both complement and extend the host specificities of existing Tn delivery vectors. Plasmids pSLX10 and pSLX23 were shown to transfer at high frequency in intergeneric matings. The lux genes which are present on each vector permit the visual monitoring of transconjugants which have retained a Tn element, but are devoid of plasmid molecules. pSLX10 and pLSX23 were efficiently used to generate a range of auxotrophic mutants in various strains of Pseudomonas as well as to clone genes from Serratia liquefaciens. These vectors may have general applicability to identify and clone genes in a wide range of Gram-negative bacteria.

Bacterial Proteins↗
PubMed1992

Vaccinia and other poxvirus expression vectors.

Over the past year improvements have been made in recombinant vaccinia virus gene expression and a new method for inserting DNA into the poxvirus genome has been developed, along with alternative methods for selecting recombinant viruses. Attenuated and non-replicating vaccinia virus and avian poxvirus vectors are now being used successfully. Field trials of an oral, wild-life rabies vaccine and phase 1 testing of human vaccines derived from vaccinia virus are underway.

Animals↗
PubMed1985

Maximizing gene expression from plasmid vectors containing the lambda PL promoter: strategies for overproducing transcription termination factor rho.

We have constructed two plasmids in which transcription of the rho gene from Escherichia coli K-12 is under the control of the lambda phage PL promoter. In p31-356, the normal rho promoter is deleted, but the remainder of the rho leader region, including the ribosome binding site, is present. In p39-AS, the rho leader is completely absent, and the lambda cII ribosome binding site replaces that of rho. Under noninducing conditions, expression of rho protein from these plasmids is repressed by the lambda cI protein in hosts carrying lambda cryptic prophage. Induction using mitomycin C or nalidixic acid in a cryptic lysogen carrying the cI+ repressor resulted in the overproduction of rho protein to levels of 3%-5% of the total cellular protein with p31-356, and to levels of approximately equal to 40% with p39-AS. The overproduced protein is functionally indistinguishable from the rho protein isolated from the K-12 strain W3110, and it can be obtained from cells harboring p39-AS in yields of up to 25 mg of rho per g of cells. In contrast to chemical induction, heat induction in four cryptic lambda lysogens carrying the thermolabile cI857 repressor failed to yield the same high levels of rho protein (with either plasmid). Our results show that chemical induction of PL-containing plasmid expression vectors can serve as a convenient and useful alternative to the commonly used method of heat induction.

Bacteriophage lambda↗
PubMed2003

Simultaneous on/off regulation of transgenes located on a mammalian chromosome with Cre-expressing adenovirus and a mutant loxP.

The site-specific recombinase Cre has often been used for on/off regulation of expression of transgenes introduced into the mammalian chromosome. However, this method is only applicable to the regulation of a single gene and cannot be used to simultaneously regulate two genes, because site-specific recombination occurs from the target loxP sequence of one regulating unit to the loxP sequence of any other unit and would eventually disrupt the structure of both regulating units. We previously reported a mutant loxP sequence with a two base substitution called loxP V (previously called loxP 2272), with which wild-type loxP cannot recombine but with which the identical mutant loxP recombines efficiently. In the present study we isolated cell lines bearing two regulating units on a chromosome containing a pair of wild-type loxP sequences or mutant loxP V sequences. After infection with Cre-expressing recombinant adenovirus AxCANCre, expression of a gene in each regulating unit was simultaneously turned on and off. Southern analyses showed that both regulating units were processed simultaneously and independently, even after infection with a limited amount of AxCANCre. The results showed that simultaneous regulation of gene expression on a mammalian chromosome mediated by Cre can be achieved by using mutant loxP V and wild-type loxP. This method may be a useful approach for conditional transgenic/knockout animals and investigation of gene function involving two genes simultaneously. Another possible application is for preparation of a new packaging cell line of viral vectors through simultaneous production of toxic viral genes.

Adenoviridae↗
PubMed1990

Ping-pong amplification of a retroviral vector achieves high-level gene expression: human growth hormone production.

Retroviral vectors offer major advantages for gene transfer studies but have not been useful for producing proteins in large quantities. This deficiency has resulted in part from interference to superinfection, which limits the numbers of active proviruses in cells. Recently, we found that these vectors amplify when they are added as calcium phosphate precipitates to cocultures of cells that package retroviruses into ecotropic and amphotropic host range envelopes. Helper-free virions from either cell type can infect the other without interference, resulting in theoretically limitless back-and-forth (ping-pong) vector replication. In initial studies, however, amplifications of a vector that contained the human growth hormone gene ceased when the hormone produced was 0.3% or less of cellular protein synthesis. This limit was caused by two factors. First, recombinant shutoff viruses that are replication defective and encode envelope glycoproteins form at a low probability during any round of the vector replication cycle and these spread in cocultures, thereby establishing interference. Single cells in shutoff cocultures therefore synthesize both ecotropic and amphotropic envelope glycoproteins, and they release promiscuous (presumably hybrid) virions. The probability of forming shutoff viruses before the vector had amplified to a high multiplicity was reduced by using small cocultures. Second, cells with large numbers of proviruses are unhealthy and their proviral expression can be unstable. Stable expresser cell clones were obtained by selection. Thereby, cell lines were readily obtained that stably produce human growth hormone as 4 to 6% of the total protein synthesis. A ping-pong retroviral vector can be used for high-level protein production in vertebrate cells.

Biotechnology↗
PubMedDate not supplied

Single-stranded DNA 'blue' T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering.

Chimeric phage-plasmid expression vectors were constructed from pUC18/19 plasmids by cloning a single-stranded DNA (ssDNA) origin of replication from bacteriophage f1 and inserting a bacteriophage T7 promoter within the beta-galactosidase gene. A T7 promoter permits in vivo or in vitro expression of single proteins by the translation of T7 RNA polymerase transcripts. Insertional inactivation of the T7 promoter-containing beta-galactosidase gene permits a simple blue-to-white color cloning assay. Compared with several helper phages that were examined, superinfection with M13K07 resulted in the highest yields of the pTZ plasmids as ssDNA viral particles. These ssDNA promoter plasmids are uniquely suited for protein engineering because they simplify cloning, oligonucleotide directed mutagenesis, verification by enzymatic sequence analysis, and expression of mutant proteins from a single vector. These vectors were utilized to eliminate an efficient transcriptional terminator of T7 RNA polymerase in the cDNA of bovine preproparathyroid hormone by oligonucleotide directed mutagenesis. The mutation changed the codon for phenylalanine-19 in the signal peptide to alanine. In a cell-free system the mutant cDNA transcripts were translated into preproparathyroid hormone, which was converted to proparathyroid hormone in the presence of microsomal membranes.

Animals↗
PubMed2002

Replication-defective genomic herpes simplex vectors: design and production.

Herpes simplex virus (HSV) may be engineered to produce flexible and efficient gene delivery vectors. Recent advances in vector design and production have built on increasing understanding of the basic biology of HSV to minimise vector toxicity and exploit viral features that give rise to lifelong latent infection in the nervous system. In addition, the emerging picture of viral cell entry has allowed early steps to be taken towards targeting viral entry to predetermined cellular subsets. Recent work has established sound principles for the straightforward production of large-scale pure preparations of vector stocks for clinical applications.

DNA, Recombinant↗
PubMed1990

Viruses as vectors.

Traditional vaccines against diseases caused by viruses are based on live attenuated viruses or killed virus preparations. Through the application of molecular biology it is now possible to consider several new approaches to making vaccines, which may combine increased efficacy with greater safety. One of these approaches is to manipulate genetically a virus so that it carries and expresses a foreign gene (or part of a gene) which codes for a protective antigen for another disease. Adeno-, polio- and herpesviruses have been engineered to act as vectors in this way but vaccinia virus remains the main candidate for a recombinant virus vector for vaccine use. The broad host-range of vaccinia virus has made it an effective vector for the analysis of expression of "foreign" antigens as well as a tool for the dissection of the host animal's immune system. For practical purposes in veterinary vaccines, recombinant viruses based on other poxviruses, with more restricted host-ranges, may have certain advantages. Work on the development of recombinant avipoxviruses and capripoxviruses as prototype vaccines for use in poultry and ruminants, respectively, is discussed and illustrated.

Animals↗
PubMed2000

Characterization of the minimal replicon of a cryptic Deinococcus radiodurans SARK plasmid and development of versatile Escherichia coli-D. radiodurans shuttle vectors.

The nucleotide sequence of a 12-kb fragment of the cryptic Deinococcus radiodurans SARK plasmid pUE10 was determined, in order to direct the development of small, versatile cloning systems for Deinococcus. Annotation of the sequence revealed 12 possible open reading frames. Among these are the repU and resU genes, the predicted products of which share similarity with replication proteins and site-specific resolvases, respectively. The products of both genes were demonstrated using an overexpression system in Escherichia coli. RepU was found to be required for replication, and ResU was found to be required for stable maintenance of pUE10 derivatives. Gel shift analysis using purified His-tagged RepU identified putative binding sites and suggested that RepU may be involved in both replication initiation and autoregulation of repU expression. In addition, a gene encoding a possible antirestriction protein was found, which was shown to be required for high transformation frequencies. The arrangement of the replication region and putative replication genes for this plasmid from D. radiodurans strain SARK is similar to that for plasmids found in Thermus but not to that for the 45.7-kb plasmid found in D. radiodurans strain R1. The minimal region required for autonomous replication in D. radiodurans was determined by sequential deletion of segments from the 12-kb fragment. The resulting minimal replicon, which consists of approximately 2.6 kb, was used for the construction of a shuttle vector for E. coli and D. radiodurans. This vector, pRAD1, is a convenient general-purpose cloning vector. In addition, pRAD1 was used to generate a promoter probe vector, and a plasmid containing lacZ and a Deinococcus promoter was shown to efficiently express LacZ.

Amino Acid Sequence↗
PubMed1995

Unaltered immunoglobulin expression in hybridoma cells modified by targeting of the heavy chain locus with an integration vector.

Chimeric antibodies against the murine T-cell antigen Thy-1.2 were generated in amounts sufficient for in vivo studies by substituting the constant gene segments via homologous recombination in the hybridoma cell. We show that an integration vector targets the heavy chain locus at high frequency even in a non-isogenic situation. Using this vector type, for the first time expression rates were obtained that were identical to the parental hybridoma. The use of the gpt selection marker seems to be crucial for efficient expression, and may overcome a recently claimed drawback of vector integration. A chimeric antibody produced by gene targeting was characterized in vitro and in vivo.

Animals↗
PubMed2003

Engineering nuclear localization signals in modular protein vehicles for gene therapy.

Amino acids from 126 to 135 of the SV40 virus T antigen act as efficient nuclear localization signal during infection but also when fused to recombinant proteins. This peptide has been inserted into two alternative acceptor sites of a modified Escherichia coli beta-galactosidase which also displays a DNA-binding domain, a cell-binding motif for integrin alpha(v)beta(3) targeting and cell internalization, and a cryptic nuclear targeting signal naturally present in the bacterial enzyme. In cultured cells, the presence of the SV40 peptide enhances the expression of a delivered DNA up to 30-fold. However, the DNA expression levels are largely depending on the chosen insertion site for the SV40 segment concomitant to the structural impact of peptide accommodation on the protein vehicle. The structural stability of the hybrid protein, apparently critical for efficient gene transfer, is discussed in the context of modular protein engineering to develop non-viral vectors for gene therapy.

Animals↗
PubMed1998

Adeno-associated virus vectors can be efficiently produced without helper virus.

The purpose of this work was to develop an efficient method for the production of adeno-associated virus (AAV) vectors in the absence of helper virus. The adenovirus regions that mediate AAV vector replication were identified and assembled into a helper plasmid. These included the VA, E2A and E4 regions. When this helper plasmid was cotransfected into 293 cells, along with plasmids encoding the AAV vector, and rep and cap genes, AAV vector was produced as efficiently as when using adenovirus infection as a source of help. CMV-driven constructs expressing the E4orf6 and the 72-M(r), E2A proteins were able to functionally replace the E4 and E2A regions, respectively. Therefore the minimum set of genes required to produce AAV helper activity equivalent to that provided by adenovirus infection consists of, or is a subset of, the following genes: the E4orf6 gene, the 72-M(r), E2A protein gene, the VA RNA genes and the E1 region. AAV vector preparations made with adenovirus and by the helper virus-free method were essentially indistinguishable with respect to particle density, particle to infectivity ratio, capsimer ratio and efficiency of muscle transduction in vivo. Only AAV vector preparations made by the helper virus-free method were not reactive with anti-adenovirus sera.

Adenoviridae↗
PubMed1999

Synthetic DNA-compacting peptides derived from human sequence enhance cationic lipid-mediated gene transfer in vitro and in vivo.

Cationic lipids can deliver genes efficiently in vitro, but are generally inhibited by the presence of serum, and their efficiency in vivo is much lower than in vitro. An attractive strategy is to induce strong DNA compaction by its association with proteins, before addition of lipids. However the use of whole proteins might present both production and immunological limitations. We have devised a system in which DNA is associated with short peptides derived from human histone or protamine, before the addition of a cationic lipid or polymer. Peptides strongly associating with DNA confer to such peptide-DNA-lipid particles an enhanced in vitro transfection efficiency over that observed with classical DNA/lipid lipoplexes, and particularly confer the capacity to transfect in the presence of serum. This acquisition of serum resistance is cell type-independent, and observed with all four lipopolyamines tested and polyethylenimine. Precompacting DNA with a histone H1-derived peptide enhances cationic lipid RPR 115335-mediated gene transfer in an in vivo model of Lewis lung carcinoma. Apart from their use in peptide-DNA-lipid association, such peptides could be useful as part of chimeric gene delivery vectors presenting a DNA-binding moiety that can be easily associated with other functional domains.

3T3 Cells↗
PubMed1997

Dicistronic tagging of genes active in embryonic stem cells of mice.

A gene-trap vector, pWH14, has been developed to tag genes expressed in embryonic stem (ES) cells of the mouse. The approach relies on the ability of the endogenous promoter to drive promoterless neo-IRES-lacZ construct producing a dicistronic mRNA consisting of the neomycin-resistance (neo) gene and the beta-galactosidase gene sequence. The neo gene produces a chimeric protein with the truncated product of the tagged gene and serves as a selectable marker for an insertion into an expressed gene. The internal ribosome entry site (IRES) sequence from murine encephalomyocarditis virus allows the translation of the second cistron, lacZ, to produce beta-galactosidase that can be used as a reporter for the expression of the tagged gene. The pWH14 vector was introduced into ES cells by electroporation, and the cells were selected for G418-resistance. About 50% of the G418-resistant colonies were stained positive for the beta-galactosidase activity. Southern analysis showed that each clone had one or more vector sequences integrated. Northern blot analysis of the clones positive for beta-galactosidase indicated that the fused RNAs containing the neo and the beta-gal genes were derived from the endogenous promoters of the tagged genes. Seven clones were chosen and injected into blastocysts, and chimeras were obtained. Two of the gene-trap insertions (wh14.1 and wh14.3) were transmitted through germ-line. In these two lines, the pattern of lacZ expression was restricted to early stages of embryos. This gene-trap vector may provide a means for tagging and studying the active genes in vivo in early embryogenesis.

Animals↗