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At least 379 records · Page 21Linked to original sources
PubMed1985

[Construction of a gene library using partial filling of DNA sticky ends].

To prepare gene libraries, the incomplete filling of protruding ends has been used. DNAs from phages EMBL 3 and EMBL 3a were sequentially digested with SalI and EcoRI, followed by addition of dTTP, dCTP, and DNA polymerase I (Klenow's fragment). Separately, a genomic DNA was partially cleaved with Sau3AI, followed by addition of dATP, dGTP, and Klenow's fragment. The fragmented phage and genomic DNAs were mixed and ligated, and the recombinant DNAs packed in vitro with the phage proteins. The effectiveness of packaging per microgram of genomic DNA was 10(5) to 10(6) (for the wild phage DNA, 10(7)). The proposed procedure is very rapid and needs only microgram quantities of genomic DNA for preparing a representative gene library. It is also useful for other vectors, containing SalI sites.

Animals↗
PubMed2002

Alphavirus vectors as tools in cancer gene therapy.

Alphavirus vectors, particularly those based on the replicon of Semliki Forest virus, have shown great potential as gene delivery vehicles for various applications in cancer gene therapy. The rapid production of high-titer recombinant SFV particles, which show impressive transduction rates in various mammalian cell lines, primary cultures and in vivo, results in high levels of transgene expression. Additionally, SFV vectors induce apoptosis in transduced host cells, which can further increase their efficiency in tumor therapy. Because of the broad host range some attempts to target the gene delivery have been engineered for Sindbis virus vectors, where IgG binding domains of protein A have been introduced into the envelope structure of the recombinant particles to allow attachment of virus to host cells through the interaction of protein A with monoclonal antibodies. SFV vectors have also been employed for the production of retrovirus-like particles for establishment of long-term gene expression. Tumor vaccine approaches have been taken by injection of SFV vectors as naked RNA molecules, DNA plasmids or recombinant particles to achieve both therapeutic and prophylactic efficacy. The continuous improvement of alphavirus vectors will further expand the application range in the future.

Alphavirus↗
PubMed1991

Strategies for improving plasmid stability in genetically modified bacteria in bioreactors.

Exploitation of recombinant organisms for the large-scale, commercial production of foreign proteins is often hampered by the problem of plasmid instability. A wide range of strategies have been reported for improving the stability of recombinant organisms. A combination of manipulating both the genetic design of recombinants and the conditions of culturing the organisms may be used to achieve stable host-vector associations during culture of recombinant organisms in bioreactors.

Bacteria↗
PubMed2001

Collagen II-pulsed antigen-presenting cells genetically modified to secrete IL-4 down-regulate collagen-induced arthritis.

We explored the possibility that pulsed antigen-presenting cells (APC) provide a model vector system for site-specific delivery of immunosuppressive proteins during collagen-induced arthritis (CIA), an animal model for rheumatoid arthritis. Thus, mice were treated with either B cells or macrophages engineered to secrete IL-4 and loaded (or not) with type II collagen (CII). Systemic injection of an IL-4-producing B cell hybridoma resulted in a reduction of arthritis severity which was further improved when APC were incubated with CII before their transfer. Unmanipulated B cells loaded with CII also exerted a potent suppressive effect. Likely, clinical amelioration was observed in mice given at priming syngeneic bone marrow-derived macrophages producing IL-4 and pulsed with CII in comparison to the other groups. When the same dose of cells was transferred at disease onset, a moderate beneficial effect was observed. Whatever the APC inoculated, the beneficial effect did not rely upon an IL-4-driven shift towards Th2 phenotype. Systemic administration of fluorescent dye labeled macrophages to arthritic mice has shown that some of these cells rapidly migrate to joints. Moreover, IL-4 transfected macrophages retained their potent capacity to present CII peptides to T cells. These findings validate the use of CII peptide-loaded engineered APC as therapeutic vector cells in CIA and allow consideration of this strategy for the administration of various anti-inflammatory proteins.

Animals↗
PubMed1999

Ten years of gene targeting: targeted mouse mutants, from vector design to phenotype analysis.

Gene targeting, defined as the introduction of site-specific modifications into the genome by homologous recombination, has revolutionarized the field of mouse genetics and allowed the analysis of diverse aspects of gene function in vivo. It is now possible to engineer specific genetic alterations ranging from subtle mutations to chromosomal rearrangements and more recently, even tissue-specific inducible gene targeting with temporo-spatial control has become feasible. This review tries to recapitulate what we have learned in this extremely rapidly expanding field during the past decade. Diverse aspects of the technique will be discussed starting from basic construct design to the analysis of complex phenotypes, including recent advances on inducible expression system. Many examples from different areas of biomedical research are given to illustrate the purpose and limitations of the employed experimental approaches.

Animals↗
PubMed1999

Lentiviruses as gene delivery vectors.

Lentivirus vectors are already used as effective gene delivery tools in cells from liver, retina, skeletal muscle and the central nervous system. In the past year, new and exciting data from gene transfer experiments in human hematopoietic progenitor cells have been obtained. Furthermore, new generations of HIV-1 based lentivirus vectors as well as new potentially less pathogenic HIV-2 based vectors have been described; however, old and new obstacles remain to be cleared.

Animals↗
PubMed2001

Efficient infection of primitive hematopoietic stem cells by modified adenovirus.

Almost all studies of adenoviral vector-mediated gene transfer have made use of the adenovirus type 5 (Ad5). Unfortunately, Ad5 has been ineffective at infecting hematopoietic progenitor cells (HPC). Chimeric Ad5/F35 vectors that have been engineered to substitute the shorter-shafted fiber protein from Ad35 can efficiently infect committed hematopoietic cells and we now show highly effective gene transfer to primitive progenitor subsets. An Ad5GFP and Ad5/F35GFP vector was added to CD34(+) and CD34(-)lineage(-) (lin(-)) HPC. Only 5-20% of CD34(+) and CD34(-)lin(-) cells expressed GFP after Ad5 exposure. In contrast, with the Ad5/F35 vector, 30-70% of the CD34(+), 50-70% of the CD34(-)lin(-) and up to 60% of the CD38(-) HPC expressed GFP and there was little evident cellular toxicity. Because of these improved results, we also analyzed the ability of Ad5/F35 virus to infect the hoechst negative 'side population' (SP) of marrow cells, which appear to be among the very earliest multipotent HPC. Between 51% and 80% of marrow SP cells expressed GFP. The infected populations retained their ability to form colonies in two short-term culture systems, with no loss of viability. We also studied the transfer and expression of immunomodulatory genes, CD40L (cell surface expression) and interleukin-2 (secreted). Both were expressed at immunomodulatory levels for >5 days. The ability of Ad5/F35 to deliver transgenes to primitive HPC with high efficiency and low toxicity in the absence of growth factors provides an improved means of studying the consequences of transient gene expression in these cells.

Adenoviridae↗
PubMed1999

Intraarterial delivery of adenovirus vectors and liposome-DNA complexes to experimental brain neoplasms.

This study investigated the intraarterial delivery of genetically engineered replication-deficient adenovirus vectors (AVs) and cationic liposome-plasmid DNA complexes (lipoDNA) to experimental brain tumors. Adenovirus or lipoDNA was injected into the internal carotid artery (ICA) of F344 rats harboring intracerebral 9L gliosarcomas, using bradykinin (BK) to selectively permeabilize the blood-tumor barrier (BTB). Brain and internal organs of the animals were collected 48 hr after vector injection and stained for expression of the marker gene product, beta-galactosidase (beta-Gal). Intracarotid delivery of AV to 9L rat gliosarcoma without BTB disruption resulted in transgene expression in 3-10% of tumor cells distributed throughout the tumor. Virus-mediated expression of beta-gal gene products in this tumor model was particularly high in small foci (< or = 0.5 mm), which had invaded the normal brain tissue surrounding the main tumor mass. In these foci more than 50% of tumor cells were transduced. BK infusion increased the amount of transgene-expressing cells in larger tumor foci to 15-30%. In the brain parenchyma only a few endothelial cells expressed beta-gal owing to AV-mediated gene transfer. Intracarotid delivery of lipoDNA bearing a cytoplasmic expression cassette rendered more than 30% of the tumor cells positive for the marker gene without BTB disruption. The pattern of distribution was in general homogeneous throughout the tumor. BK infusion was able to increase further the number of transduced tumor cells to more than 50%. Although lipoDNA-mediated gene transfer showed increased efficacy as compared with AV-mediated gene transfer, it had less specificity since a larger number of endothelial and glial cells also expressed the transgene. AV and lipoDNA injections, in the absence and presence of BK, also resulted in transduction of peripheral organs. AV showed its known predilection for liver and lung. In the case of lipoDNA, parenchymal organs such as liver, lung, testes, lymphatic nodes, and especially spleen, were transduced. These findings indicate that intracarotid application of AV and lipoDNA vectors can effectively transduce tumor cells in the brain, and that BTB modulation by BK infusion can further increase the number of transgene-expressing tumor cells.

Adenoviridae↗
PubMed1996

Engineering a de novo-designed coiled-coil heterodimerization domain off the rapid detection, purification and characterization of recombinantly expressed peptides and proteins.

Using the techniques of genetic engineering and the principles of protein de novo design, we have developed a unique affinity matrix protein tag system as a rapid, convenient and sensitive method to detect, purify and characterize newly expressed recombinant peptides or proteins from cell extracts. The method utilizes two de novo-designed linear peptide sequences that can selectively dimerize to form the stable protein motif, the two-stranded alpha-helical coiled-coil. In this method, a recombinant bacterial expression vector pRLDE has been engineered so that one of the dimerization strands (E-coil) is expressed as a C-terminal fusion tag on newly expressed peptides or proteins, while the other (K-coil) is either biotin-labeled for detection in a Western blot-type format or immobilized on an insoluble silica support for selective dimerization affinity chromatography. Recombinantly expressed peptides from Escherichia coli containing the dimerization tag have been produced, detected and purified using this method. The recombinant peptides were easily and clearly identified using the biotin-labeled coil, while the single-step affinity purification results indicated the purity of the affinity purified expressed peptides to be > 95%, as assessed by reversed-phase chromatography. The stability of the dimerization domain also allows for the purified peptide to be left attached to the matrix, thus creating a new peptide-bound column that can be used to study peptide-protein or peptide-ligand interactions. Therefore this system offers a new alternative to existing peptide or protein fusion tags and demonstrates the utility of a de novo-designed system.

Amino Acid Sequence↗
PubMed2001

Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis.

Adeno-associated virus (AAV) vectors package single-stranded genomes and require host-cell synthesis of the complementary strand for transduction. However, when the genome is half wild-type size, AAV can package either two copies, or dimeric inverted repeat DNA molecules. Dimeric, or self-complementary molecules (scAAV) should spontaneously reanneal, alleviating the requirement for host-cell DNA synthesis. We generated and characterized scAAV vectors in order to bypass the rate-limiting step of second-strand synthesis. In vitro, scAAV vectors were five- to 140-fold more efficient transducing agents than conventional rAAV, with a 5.9:1 particle to transducing unit ratio. This efficiency is neither greatly increased by co-infection with Ad, nor inhibited by hydroxyurea, demonstrating that transduction is independent of DNA synthesis. In vivo, scAAV expressing erythropoietin resulted in rapid and higher levels of hematocrit than a conventional single-stranded vector. These novel scAAV vectors represent a biochemical intermediate in rAAV transduction and should provide new insights into the biology of vector transduction.

Animals↗
PubMed1994

Structures of replacement vectors for efficient gene targeting.

The relationship between DNA structure of replacement vectors and gene targeting efficiency was studied using positive-negative selection. The vectors contained pBR322 DNA, a bacterial neomycin-resistance gene (neo) for positive selection, a herpes simplex virus (HSV) thymidine kinase gene (tk) for negative selection, and a mouse genomic fragment, including exons 1 to 3 of the transthyretin (ttr) gene. The neo gene that confers G418 resistance was inserted into the second ttr exon, and the HSV-tk gene that confers gancyclovir (GANC) sensitivity was added to the 3' end of the ttr fragment. The vectors were linearized by digesting with restriction enzyme(s) and transfected into mouse embryonal carcinoma F9 cells. In this system, the enrichment by GANC selection as well as the frequency of gene targeting was increased by placing the pBR322 DNA at the 3' end of the HSV-tk gene. Adding one more HSV-tk gene at the 5' end of the ttr fragment did not increase the enrichment by GANC selection. This enrichment factor was also increased by reducing the size of the ttr fragment present between the two selection markers. However, it decreased the frequency of gene targeting and, overall, it did not increase the efficiency of isolating targeted clones. When structures of the vector DNA fragments present in 20 G418-resistant and GANC-resistant non-targeted clones were examined by Southern blot analysis, the inefficiency of GANC selection proved to be mostly caused by exonucleolytic degradation of HSV-tk genes progressing from ends of the vectors.

Animals↗
PubMed1992

Introducing mutations into the single-copy chromosomal 23S rRNA gene of the archaeon Halobacterium halobium by using an rRNA operon-based transformation system.

A vector-transformation system is described that permits replacement of a portion of the single rRNA operon of the archaeon Halobacterium halobium with a homologous fragment from a vector-borne gene. The vector construct contains three functional sections: (i) an entire H. halobium rRNA operon with two selective mutations in the 23S rRNA gene, the substitutions of A----G at position 1159 conferring resistance to thiostrepton and C----U at position 2471 conferring resistance to anisomycin; (ii) the complete pHSB1 plasmid from Halobacterium sp. SB3, which interferes with vector maintenance in the transformed halobacterial cells; and (iii) a segment of the pBR322 plasmid that permits vector replication in Escherichia coli. Transformation of H. halobium with the vector plasmid generates cells resistant to both anisomycin and thiostrepton that can be selected for, and discriminated from spontaneous mutants, by a two-step selection procedure. After transformation, the plasmid recombines homologously with the chromosome so that the plasmid-borne rDNA segment with resistance markers substitutes for the corresponding region of the chromosomal rRNA operon, and the transforming plasmid is lost. Eventually, this leads to a homogeneous population of the mutant ribosomes in the cell. Other mutations that are engineered in the vector-borne rRNA sequences can be transferred to the chromosomal rRNA operon concomitantly with the selective markers. The system has considerable potential for ribosomal engineering.

Anisomycin↗
PubMed1986

Shuttle mutagenesis: a method of transposon mutagenesis for Saccharomyces cerevisiae.

We have extended the method of transposon mutagenesis to the eukaryote, Saccharomyces cerevisiae. A bacterial transposon containing a selectable yeast gene can be transposed into a cloned fragment of yeast DNA in Escherichia coli, and the transposon insertion can be returned to the yeast genome by homologous recombination. Initially, the cloned yeast DNA fragment to be mutagenized was transformed into an E. coli strain containing an F factor derivative carrying the transposable element. The culture was grown to allow transposition and cointegrate formation and, upon conjugation, recipients were selected that contained yeast sequences with transposon insertions. The yeast DNA was removed from the vector by restriction endonuclease digestion, and the transposon insertion was transformed into yeast. The procedure required a minimum number of manipulations, and each transconjugant colony contained an independent insertion. We describe 12 transposon Tn3 derivatives for this procedure as well as several cloning vectors to facilitate the method.

Cloning, Molecular↗
PubMed1998

DNA vaccines: vector design, delivery, and antigen presentation.

Inoculations with antigen-expressing plasmid DNAs (DNA vaccines) in the production of protective immune responses. Since the initial development of DNA vaccines more than 5 years ago, major strides have been made in the design of efficient vaccine vectors and in the process of vaccine delivery. However, many questions remain regarding the mechanism of cellular transfection and in the development of immune responses. This review addresses functional aspects of DNA vaccines, including vector design and delivery, as well as cellular transfection and antigen presentation.

Animals↗
PubMed1998

pTRIDENT, a novel vector family for tricistronic gene expression in mammalian cells.

We constructed tricistronic expression vectors for the simultaneous and coordinated expression of three independent genes in mammalian cells. One single promoter allows high level and, in some vectors, adjustable transcription of all three cistrons. Whereas the first cistron is translated in a cap-dependent manner, the subsequent ones utilize intercistronic regions of viral origin such as the internal ribosomal entry site of poliovirus or the cap-independent translation enhancer of encephalomyocarditis virus for enhanced translation. Three multiple cloning sites with a total of up to 18 unique restriction sites allow sequential cloning of the genes of interest. The modular structure of this pBluescript(R)-based high copy number vector system allows straightforward movement of individual cistrons among members of the pTRIDENT family, and facilitates their combination with existing expression vectors.

Animals↗