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At least 451 records · Page 25Linked to original sources
PubMed2001

Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria.

Mobile group II introns can be retargeted to insert into virtually any desired DNA target. Here we show that retargeted group II introns can be used for highly specific chromosomal gene disruption in Escherichia coli and other bacteria at frequencies of 0.1-22%. Furthermore, the introns can be used to introduce targeted chromosomal breaks, which can be repaired by transformation with a homologous DNA fragment, enabling the introduction of point mutations. Because of their wide host range, mobile group II introns should be useful for genetic engineering and functional genomics in a wide variety of bacteria.

Base Sequence↗
PubMed2002

Characterization of a laboratory-derived, high-level ampicillin-resistant Salmonella enterica serovar Typhimurium strain that caused meningitis in an infant.

A Salmonella enterica serovar Typhimurium strain that harbored a plasmid carrying a TEM-1-type beta-lactamase gene was isolated from the blood and cerebrospinal fluid of an infant with meningitis. This 3.2-kb plasmid was further characterized to be a nonconjugative pGEM series cloning vector containing a foreign insert. The strain was likely laboratory derived and contaminated the environment before it caused the infection.

Ampicillin↗
PubMed2001

Human immunodeficiency virus type 2 lentiviral vectors: packaging signal and splice donor in expression and encapsidation.

Retroviral vectors provide the means for gene transfer with long-term expression. The lentivirus subgroup of retroviruses, such as human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2), possesses a number of regulatory and accessory genes and other special elements. These features can be exploited to design vectors for transducing non-dividing as well as dividing cells with the potential for regulated transgene expression. Encapsidation of the transgene RNA in lentiviral vectors is determined by the leader sequence-based multipartite packaging signal. Embedded in the packaging signal is a major splice donor site that, this study shows, is not by itself essential for transgene expression or encapsidation. We designed HIV-2 vectors that contained all the sequence elements thought to be necessary and sufficient for vector RNA encapsidation. Unexpectedly, despite abundant expression, only a small fraction of the transgene RNA was encapsidated and the titre of the vector was low. Redesign of the vector with a mutant splice donor resulted in increased vector RNA encapsidation and yielded vectors with high titre. Inefficient encapsidation by the conventionally designed vector was not due to suboptimal Rev responsive element (RRE)-Rev function. Varying the length of RRE in the vector did not change vector RNA encapsidation, nor did the introduction of a synthetic intron into the mutant vector. The vector RNA with the intact splice donor may have been excessively spliced, decreasing the amount of packageable RNA. A titre of 10(5) transducing units (TU)/ml was readily obtained for vectors with the neo or GFP transgene, and the vector could be concentrated to a titre of 1-5x10(7) TU/ml.

Capsid↗
PubMed2000

Adenoviral vector design for high-level transgene expression in primitive human hematopoietic progenitors.

Adenoviral vector-mediated transient gene expression can provide new possibilities for ex vivo manipulation of quiescent hematopoietic stem cells (HSC). In order to define a suitable expression cassette for high levels of transgene expression in HSCs, we have studied the level of transgene expression in human CD34+CD38- cells using adenoviral vectors with various gene expression cassettes encoding the enhanced green fluorescence protein (EGFP) gene. CD34+ hematopoietic cells were cultured in serum-free medium with megakaryocyte growth and development factor (MGDF) alone for supporting the survival of primitive progenitors or with MGDF, c-kit ligand (KL) and flt3 ligand (FL) for inducing proliferation of primitive progenitors. With all the vectors tested, higher percentages of EGFP expressing cells were found in CD34+CD38- cells than those in CD34+CD38high cells from all donors tested. The phosphoglycerate kinase (PGK)-1 promoter was found to allow higher levels of EGFP expression than the human cytomegalovirus (HCMV) promoter in CD34+CD38- cells. Replacing the SV40 polyadenylation signal with the human beta-globin gene IVS2 and polyadenylation signal in the expression cassette (Ad5xPGK-EGFP-beta-globin) enhanced the level of EGFP expression markedly further. These results provide a guideline for the development of adenoviral vectors for gene expression in human primitive hematopoietic progenitor cells. Gene Therapy (2000) 7, 2132-2138.

ADP-ribosyl Cyclase↗
PubMed2001

A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus.

Fully deleted adenovirus vectors (FD-AdVs) would appear to be promising tools for gene therapy. Since these vectors are deleted of all adenoviral genes, they require a helper adenovirus for their propagation. The contamination of the vector preparation by the helper limits the utility of currently existing FD-AdVs in gene therapy applications. We have developed an alternative system for the propagation of FD-AdVs, in which the adenoviral genes essential for replication and packaging of the vector are delivered into producer cells by a baculovirus-adenovirus hybrid. A hybrid baculovirus Bac-B4 was constructed to carry a Cre recombinase-excisable copy of the packaging-deficient adenovirus genome. Although the total size of the DNA insert in Bac-B4 was 38 kb, the genetic structure of this recombinant baculovirus was stable. Bac-B4 gave high yields in Sf9 insect cells, with titers of 5 x 10(8)p.f.u./ml before concentration. Transfection of 293-Cre cells with lacZ-expressing FD-AdV plasmid DNA followed by infection by Bac-B4 at a MOI of 2000 p.f.u./ml resulted in rescue of the helper-free vector. Subsequent passaging of the obtained FD-AdV using Bac-B4 as a helper resulted in approximately 100-fold increases of the vector titer at each passage. This resulting vector was completely free of helper virus and was able to transduce cultured 293 cells. However, scaling-up of FD-AdV production was prevented by the eventual emergence of replication-competent adenovirus (RCA). Experiments are underway to optimize this system for the large-scale production of helper virus-free FD-AdVs and to minimize the possibility of generation of replication-competent adenovirus (RCA) during vector production. This baculovirus-based system will be a very useful alternative to current methods for the production of FD-AdVs.

Adenoviridae↗
PubMed1991

Testing an "in-out" targeting procedure for making subtle genomic modifications in mouse embryonic stem cells.

We have introduced a 4-bp insertion into the hypoxanthine phosphoribosyltransferase (HPRT) gene of a mouse embryonic stem (ES) cell line by using an "in-out" targeting procedure. During the in step, a homologous integration reaction, we targeted a correcting plasmid to a partially deleted hprt- locus by using an integrating vector that carried a 4-bp insertion in the region of DNA homologous to the target locus. HPRT+ recombinants were isolated by direct selection in hypoxanthine-aminopterin-thymidine (HAT) medium. The HATr cell lines were then grown in medium containing 6-thioguanine (6-TG) to select for hprt- revertants resulting from the excision of the integrated vector sequences. The revertants were examined by Southern blot hybridization to determine the accuracy of this out reaction and the frequency of retaining the 4-bp modification in the genome. Of the 6-TGr colonies examined, 88% had accurately excised the integrated vector sequences; 19 of 20 accurate revertants retained the 4-bp insertion in the resulting hprt- gene. We suggest a scheme for making the in-out targeting procedure generally useful to modify the mammalian genome.

Animals↗
PubMed1988

An EBV-based mammalian cell expression vector for efficient expression of cloned coding sequences.

The construction of a mammalian cell expression vector using human cytomegalovirus immediate early gene enhancer to initiate transcription of inserted coding sequences is described. The vector also carries Epstein-Barr virus EBNA-1 nuclear antigen gene, ori-P sequences and hygromycin B resistance gene hph from E. coli. The expression capacity of this construct was tested by inserting the chloramphenicol acetyltransferase (CAT) gene into the vector. The EBV-CAT construct was transfected into various cell lines and high levels of CAT activity were obtained in human and monkey cells. In these cells, the vector DNA also replicates as an extrachromosomal element having 1 to 20 copies per cell. In most cases, the vector copy number and the expression level of inserted gene was in positive correlation in different cell clones.

Acetyltransferases↗
PubMed1995

Genetic approaches to the study of cytokine regulation of mucosal immunity.

Efforts to design effective mucosal vaccines have been hampered by an incomplete understanding of factors controlling the development of mucosal immunity. It is now clear, however, that T cell-derived cytokines play a major role. Recent developments in 'gene knockout' technology have allowed the generation of strains of mice in which particular genes have been inactivated. The availability of mice rendered deficient for production of Th2 cytokines has facilitated studies of the induction and development of mucosal immune responses in the absence of these factors. We have used several genetic approaches, including cytokine-deficient mice and recombinant vectors constructed to express genes for a range of different cytokines, to demonstrate the importance of these factors in the mucosa. Such genetic approaches appear to represent powerful tools for in vivo studies of the influence of cytokines in mucosal immunoregulation.

Animals↗
PubMed1987

Construction of expression vectors for the production of interferons in yeast.

Expression vectors designed for the production of foreign proteins in yeast are constructed as a composite of functional DNA segments brought together in a single plasmid DNA molecule. Such a composite DNA molecule constitutes a complex entity in terms of its replication and selection functions (in both E. coli and S. cerevisiae) and its gene expression properties. The latter depend critically upon transcriptional control signals (the promoter and terminator regions from a highly expressed yeast gene) that are used to direct the expression of foreign genes in yeast cells. This paper describes the construction of such an expression vector based on the transcription control signals of the yeast phosphoglycerate kinase gene and its application to the production of human interferon-alpha's in yeast cells.

Cloning, Molecular↗
PubMed1994

Gene targeting by a vector with hairpin-shaped oligonucleotide caps.

To improve the efficiency of gene targeting, a linearized vector containing part of the mouse transthyretin (ttr) gene, a bacterial neomycin-resistance gene and a herpes simplex virus thymidine kinase gene was capped with hairpin-shaped oligonucleotides. The capped vector not only improved the efficiency of positive-negative selection about 3.5-fold, but also increased about 2.5-fold the ratio of ttr gene targeted clones to the selectants.

Animals↗
PubMed1988

Expression of human calmodulin cDNA in Escherichia coli and characterization of the protein.

A cDNA clone of human calmodulin, isolated from liver, was subcloned into the expression vector pKK233-2. The resulting expression plasmid, designated pCWCaM1, produced human calmodulin in Escherichia coli SG5. The cDNA was sequenced using novel primers designed for use in plasmid-sequencing protocols with pKK233-2 and pKK223-3. The expressed calmodulin was purified and subjected to NMR analysis which revealed a structure essentially the same as natural calmodulin isolated from human tissue. The activation of myosin light chain kinase by the genetically engineered human calmodulin and bovine brain calmodulin was studied and found to be comparable to a high degree. The expressed calmodulin appears to be comparable to normal calmodulin and can be used for site-directed mutagenesis and structure/function investigations.

Base Sequence↗
PubMed1999

Escherichia coli skp chaperone coexpression improves solubility and phage display of single-chain antibody fragments.

Expression of single-chain antibody fragments (scAb)in the periplasm of Escherichia coli often results in low soluble product yield and cell lysis. We have increased scAb solubility and prevented cell culture lysis by coexpressing the E. coli Skp chaperone gene. A mutant Skp cistron was linked to a bacteriophage T7 gene 10 translational initiation region and placed either downstream of a scAb gene within an isopropyl beta-d-thiogalactopyranoside-inducible expression cassette or on a separate colE1-compatible arabinose-inducible vector. Increases in scAb solubility reflected the amount of coexpressed Skp. A bacteriophage display vector that was also engineered to coexpress Skp permitted display of a virtually undisplayable scAb and should prove useful in expanding library sizes.

Bacterial Proteins↗
PubMed2003

Generation of an adenoviral vector containing an addition of a heterologous ligand to the serotype 3 fiber knob.

As an initial assessment of the feasibility of employing the adenovirus serotype 3 (Ad3) fiber knob as a locale for introducing a tropism-modifying motif, we generated an adenoviral vector containing a six-histidine tag genetically fused to the carboxy-terminus of the Ad3 fiber knob. The heterologous tag proved to be accessible for binding in the context of the virion and, moreover, had rendered the modified vector capable of mediating gene transfer through an artificial, non-Ad3 receptor.

Adenoviridae↗
PubMed2000

Insulation from viral transcriptional regulatory elements improves inducible transgene expression from adenovirus vectors in vitro and in vivo.

Recombinant adenoviruses (Ad) are attractive vectors for gene transfer in vitro and in vivo. However, the widely used E1-deleted vectors as well as newer generation vectors contain viral sequences, including transcriptional elements for viral gene expression. These viral regulatory elements can interfere with heterologous promoters used to drive transgene expression and may impair tissue-specific or inducible transgene expression. This study demonstrates that the activity of a metal-inducible promoter is affected by Ad sequences both upstream and downstream of the transgene cassette in both orientations. Interference with expression from the heterologous promoter was particularly strong by viral regulatory elements located within Ad sequences nucleotides 1-341. This region is present in all recombinant Ad vectors, including helper-dependent vectors. An insulator element derived from the chicken gamma-globin locus (HS-4) was employed to shield the inducible promoter from viral enhancers as tested after gene transfer with first-generation Ad vectors in vitro and in vivo. Optimal shielding was obtained when the transgene expression cassette was flanked on both sides by HS-4 elements, except for when the HS-4 element was placed in 3'-->5' orientation in front of the promoter. The insulators reduced basal expression to barely detectable levels in the non-induced stage, and allowed for induction factors of approximately 40 and approximately 230 in vitro and in vivo, respectively. Induction ratios from Ad vectors without insulators were approximately 40-fold lower in vitro and approximately 15-fold lower in vivo. This study proves the potential of insulators to improve inducible or tissue-specific gene expression from adenovirus vectors, which is important for studying gene functions as well as for gene therapy approaches. Furthermore, our data show that insulators exert enhancer-blocking effects in episomal DNA.

Adenoviridae↗
PubMed1987

Construction of a defective retrovirus containing the human hypoxanthine phosphoribosyltransferase cDNA and its expression in cultured cells and mouse bone marrow.

Defective ecotropic and amphotropic retroviral vectors containing the cDNA for human hypoxanthine phosphoribosyltransferase (HPRT) were developed for efficient gene transfer and high-level cellular expression of HPRT. Helper cell clones which produced a high viral titer were generated by a simplified method which minimizes cell culture. We used the pZIP-NeoSV(X) vector containing a human hprt cDNA. Viral titers (1 X 10(3) to 5 X 10(4)/ml) of defective SVX HPRT B, a vector containing both the hprt and neo genes, were increased 3- to 10-fold by cocultivation of the ecotropic psi 2 and amphotropic PA-12 helper cells. Higher viral titers (8 X 10(5) to 7.5 X 10(6] were obtained when nonproducer NIH 3T3 cells or psi 2 cells carrying a single copy of SVX HPRT B were either transfected or infected by Moloney leukemia virus. The SVX HPRT B defective virus partially corrected the HPRT deficiency (4 to 56% of normal) of cultured rodent and human Lesch-Nyhan cells. However, instability of HPRT expression was detected in several infected clones. In these unstable variants, both retention and loss of the SVX HPRT B sequences were observed. In the former category, cells which became HPRT- (6-thioguanine resistant [6TGr]) also became G418s, indicative of a cis-acting down regulation of expression. Both hypoxanthine-aminopterin-thymidine resistance (HATr) and G418r could be regained by counterselection in hypoxanthine-aminopterin-thymidine. In vitro mouse bone marrow experiments indicated low-level expression of the neo gene in in vitro CFU assays. Individual CFU were isolated and pooled, and the human hprt gene was shown to be expressed. These studies demonstrated the applicability of vectors like SVX HPRT B for high-titer production of defective retroviruses required for hematopoietic gene transfer and expression.

Bone Marrow↗
PubMed1999

Cyclosporin A reduces the inflammatory response to a multi-mutant herpes simplex virus type-1 leading to improved transgene expression in sympathetic preganglionic neurons in hamsters.

Herpes simplex virus type 1 (HSV-1) based vectors hold great promise for gene transfer to CNS neurons. Problems such as loss of transgene expression, vector-associated cytotoxicity and the immune response to the vector or encoded transgene still remain obstacles to success. We used a replication-defective, HSV-1 vector (14Hdelta3vhsZ) that was engineered to have reduced cytotoxicity and express recombinant beta-galactosidase. A previous study in our laboratory showed no evidence for cytotoxicity in infected neurons although an inflammatory infiltrate occurred around infected cells and transgene expression was lost between 5 and 8 days. The immune response consisted of a primary response at the site of inoculation (adrenal gland), and a secondary immune response in the spinal cord around infected adrenal sympathetic preganglionic neurons due to retrograde transport of the vector. We tested whether conventional immunosuppressants could reduce the secondary immune response, leading to improved transgene expression at the secondary CNS site. 14Hdelta3vhsZ was injected into the adrenal gland in hamsters 1 day after immunosuppressant treatment began. Non-drug treated, 14Hdelta3vhzZ-infected hamsters were used as controls. Cyclosporin A administration led to the most persistent beta-galactosidase activity in neurons at 5 and 8 days. Methylprednisolone treatment resulted in the greatest reduction in the inflammatory cell infiltrate but the numbers of infected neurons did not increase concomitantly. This suggested no direct relationship between extent of the inflammatory cell infiltrate and level of transgene expression. These data demonstrate the potential of cyclosporin A as an immunosuppressant adjunct treatment for HSV-1 vector-mediated gene transfer from a peripheral site to neurons in the spinal cord.

Adrenal Glands↗
PubMed1987

Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template.

The important features of the protocol described here are as follows: First, the procedure consists of a few simple steps and results in a reasonably high frequency of mutagenesis. Second, using two primers, there is no need to isolate covalently closed double-stranded molecules as in our previous method. Third, the use of vectors derived from single-stranded phage facilitates template preparation, mutagenesis efficiency, screening, and DNA sequencing. Fourth, the same basic steps can be directly applied when using the single-stranded pUC derivatives.

Base Sequence↗
PubMed1998

The expression of the protective antigen of Bacillus anthracis in Bacillus subtilis.

The expression of Bacillus anthracis protective antigen (PA) in B. subtilis from the pag gene in pPA101-1 was explored in different genetic backgrounds in an attempt to identify opportunities to maximize expression. Introduction of AtxA, which positively regulates PA expression in B. anthracis did not improve expression levels in the protease-deficient strain WB600. Plasmid pPA101-1 was found to carry a deletion which created a new fusion point between vector and insert sequence, and which removed part of the AtxA binding site. The deletion may have occurred as a consequence of recombination between TCTAT sequences within both the vector and insert. Host mutations could influence expression; PA levels from pPA101-1 are threefold higher in a ccpA mutant than in an otherwise isogenic parent, and eightfold higher in an abrB mutant. These data demonstrate that the introduction of mutations affecting catabolite repression and growth phase regulation results in an increase in the yield of PA in this host-vector system. Combining these mutations with a multiply protease-negative background could potentially allow further improvements in PA yield.

Antigens, Bacterial↗