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At least 757 records · Page 42Linked to original sources
PubMed2004

A resource for large-scale RNA-interference-based screens in mammals.

Gene silencing by RNA interference (RNAi) in mammalian cells using small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) has become a valuable genetic tool. Here, we report the construction and application of a shRNA expression library targeting 9,610 human and 5,563 mouse genes. This library is presently composed of about 28,000 sequence-verified shRNA expression cassettes contained within multi-functional vectors, which permit shRNA cassettes to be packaged in retroviruses, tracked in mixed cell populations by means of DNA 'bar codes', and shuttled to customized vectors by bacterial mating. In order to validate the library, we used a genetic screen designed to report defects in human proteasome function. Our results suggest that our large-scale RNAi library can be used in specific, genetic applications in mammals, and will become a valuable resource for gene analysis and discovery.

Animals↗
PubMed2003

Virus-induced gene silencing in plants.

Virus-induced gene silencing (VIGS) is a technology that exploits an RNA-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying inserts derived from host genes the process can be additionally targeted against the corresponding mRNAs. VIGS has been used widely in plants for analysis of gene function and has been adapted for high-throughput functional genomics. Until now most applications of VIGS have been in Nicotiana benthamiana. However, new vector systems and methods are being developed that could be used in other plants, including Arabidopsis. Here we discuss practical and theoretical issues that are specific to VIGS rather than other gene "knock down" or "knockout" approaches to gene function. We also describe currently used protocols that have allowed us to apply VIGS to the identification of genes required for disease resistance in plants. These methods and the underlying general principles also apply when VIGS is used in the analysis of other aspects of plant biology.

Gene Silencing↗
PubMed1984

High-efficiency gene transfer into mammalian cells: generation of helper-free recombinant retrovirus with broad mammalian host range.

We have constructed a chimeric retrovirus genome containing ecotropic gag-pol sequences from Moloney murine leukemia virus and envelope sequences derived from the amphotropic virus 4070A. This reconstructed genome, termed pMAV-psi-, lacks the psi site required for encapsidation of the viral genome. NIH 3T3 cells transfected with pMAV-psi-, called psi-AM lines, are capable of producing high titer stocks of helper-free recombinant retrovirus with amphotropic host range after transfection with recombinant retroviral vectors carrying the neomycin phosphotransferase gene. Most transfected psi-AM cells remain helper-free, even after months in culture. psi-AM virus stocks infect nearly all human and murine cell lines tested thus far, as assayed by resistance to the neomycin analogue G418. Southern and RNA blot analyses of psi-AM-infected human cells show that recombinant murine retroviruses integrate randomly into genomic DNA as normal proviruses and express high levels of the subgenomic and genomic viral messages in the expected stoichiometry of 1:1.

Animals↗
PubMed2003

A novel neurotropic expression vector based on the avirulent A7(74) strain of Semliki Forest virus.

Semliki Forest virus (SFV), an enveloped alphavirus of the family Togaviridae, infects a wide range of mammalian host cells. Most strains are neurotropic but differ in virulence. The authors took advantage of the nonpathogenic properties of SFV strain A7(74), cloned recently in their laboratory, and constructed a replication-proficient expression vector to target the central nervous system (CNS) for heterologous gene expression. The vector, termed VA7, was engineered to drive expression of foreign inserts through a second subgenomic promoter inserted in the viral 3' nontranslated region (NTR). Infectious virus was obtained by in vitro transcription and transfection into BHK cells, and was shown to direct synthesis of heterologous proteins in several mammalian cell lines. Although novel expression vehicle is not applicable for targeting specific cell populations within the CNS in its present form, in cultured rat hippocampal slices, VA7 encoding enhanced green fluorescent protein (EGFP) efficiently transduced pyramidal cells, interneurons, and glial cells. With prolonged time post infection, the number of EGFP-expressing neurons in hippocampal slices increased. Mice infected intraperitoneally with the recombinant virus remained completely asymptomatic but showed CNS expression of EGFP as evidenced by immunohistochemistry. SFV A7(74) is a nonintegrating virus, which gives rise to a randomly distributed, patchy infection of the adult CNS that is cleared within 10 days. With the advantage of noninvasive administration, the expression vector described in this work is thus applicable for short-term gene expression in the CNS.

Animals↗
PubMed2003

Validating the hen as a bioreactor for the production of exogenous proteins in egg white.

Increased demand for the production of human biopharmaceuticals in transgenic organisms has led to an intensive effort to develop the hen as a bioreactor producing exogenous proteins in egg white via transgenesis. To date, however, robust methods for transgenic modification of the avian genome have been lacking. We have used a replication-defective retroviral vector derived from avian leukosis virus (ALV) to generate transgenic chickens expressing bacterial beta-lactamase secreted into serum and egg whites through several generations. Expression was driven by the ubiquitous cytomegalovirus (CMV) promoter. Here we describe results from a transgenic lineage (Harvey et al., 2002a,b) in which (1) the transgene was stably transmitted from a G1 founder male (5657) through several generations without silencing, (2) the protein was biologically active, and (3) the level of expression in egg whites was doubled in a G3 homozygote.

Animals↗
PubMed2003

Specific immunotherapy of experimental myasthenia by genetically engineered APCs: the "guided missile" strategy.

Although treatment of MG with general immunosuppressive agents is often effective, it has important drawbacks, including suppression of the immune system as a whole, with the risks of infection and neoplasia, and numerous other adverse side effects. Ideally, treatment of MG should eliminate the specific pathogenic autoimmune response to AChR, without otherwise suppressing the immune system or producing other adverse side effects. Although antibodies to AChR are directly responsible for the loss of AChRs at neuromuscular junctions in MG, the AChR antibody response is T cell-dependent, and immunotherapy directed at T cells can abrogate the autoantibody response, with resulting benefit. As in other autoimmune diseases, the T cell response in MG is highly heterogeneous. The design of specific immunotherapy must take this heterogeneity into account and target the entire repertoire of AChR-specific T cells. We describe our investigation of a novel strategy for specific immunotherapy of MG, involving gene transfer to convert antigen-presenting cells (APCs) to "guided missiles" that target AChR-specific T cells, and that induce apoptosis and elimination of those T cells. This strategy uses the ability of APCs from a given individual to present the entire spectrum of AChR epitopes unique for that individual, and thereby to target the entire repertoire of antigen-specific T cells of the same individual. Using viral vectors, we have genetically engineered the APCs to process and present the most important domain of the AChR molecule, and to express a "warhead" of Fas ligand (FasL) to eliminate the activated AChR-specific T cells with which they interact. Our results show that the APCs express the appropriate gene products, and effectively and specifically eliminate AChR-specific T cells by the Fas/FasL pathway, while sparing T cells of other specificities.

Animals↗
PubMed2001

Efficient FLP recombination in mouse ES cells and oocytes.

We report an improved vector, pCAGGS-FLPe, for transient expression of the enhanced FLP recombinase in mouse ES cells and oocytes. In standard transfection experiments, about 6% of total ES colonies showed FLP recombination, albeit with mosaicism within each colony. After microinjection of pCAGGS-FLPe into oocytes, about one-third of heterozygotic mice born showed complete FLP recombination. Thus pCAGGS-FLPe presents two practical options for removal of FRT cassettes in mice.

Amino Acid Sequence↗
PubMed2000

Comparison of two kinds of methods to determine the titer of recombinant retrovirus containing beta-globin gene based on G418 selection.

Four recombinant retrovirus (RV) vectors containing human beta-globin gene and regulatory elements were constructed. To determine the titers of recombinant RV from corresponding producer cell lines, we compared two kinds of method (the simple and the conventional) based on G418 resistance, which is conferred by neo gene of RV vector. The results demonstrated that the simple method shortened the selection period to 3 d instead of 10-12 d with the conventional method and reduced the amount of work; importantly, the titers determined by the simple method were not different significantly from those measured by the conventional method. It can be concluded that the simple method can be used to determine the titers of recombinant RV containing not only cDNA but also genomic DNA with introns and complex regulatory elements instead of the conventional method.

3T3 Cells↗
PubMed1996

Construction and screening of M13 phage libraries displaying long random peptides.

We have constructed two phage display libraries expressing N-terminal pIII fusions in M13 composed of 37 and 43 random amino acid domains, respectively. The D38 library expresses 37 random amino acids with a central alanine residue, and the DC43 library contains 43 random amino acids with a central cysteine flanked by two glycine residues, giving the displayed peptide the potential to form disulfide loops of various sizes. We demonstrate that the majority of random sequences in both libraries are compatible in pentavalent display with phage viability. The M13 phage display vector itself has been engineered to contain a factor Xa protease cleavage site to provide an alternative to acid elution during affinity selection. An in-frame amber mutation has been inserted between the pIII cloning sites to allow for efficient selection against nonrecombinant phage in the library. These libraries have been panned against mAb 7E11-C5, which recognizes the prostate-specific membrane antigen (PSM). Isolated phage display a consensus sequence that is homologous to a region in the PSM molecule.

Amino Acid Sequence↗
PubMed1996

Separation of E. coli expressing functional cell-wall bound antibody fragments by FACS.

BACKGROUND: The rapid development of recombinant antibody technology in the last few years has facilitated the generation of antibody libraries in bacteria. Recombinant antibodies against various antigens have been selected from these libraries by presenting each antibody on the surface of a phagemid particle that contains the antibody's gene. An alternative screening system is the display of antibody fragments on bacteria. A major advantage is the possibility to select single cells directly from a large number of bacteria by using fluorescently labeled antigens and fluorescence assisted cell sorting (FACS). OBJECTIVES: pAP is an expression vector for the bacterial display of antibody fragments. E. coli transformed with pAP express a single chain antibody (scFv) fused to the peptidoglycan-associated-lipoprotein (PAL). This fusion protein binds strongly to the cell wall. To employ this system for screening, we have investigated the possibility of selecting antigen-specific clones by FACS. STUDY DESIGN AND RESULTS: Several DNA fragments coding for various scFvs were inserted into the pAP expression vector. E. coli were transformed with these plasmids and immunostained with fluorescent antigens under given conditions. We were able to select stained E. coli expressing a specific scFv from unstained E. coli expressing a non-binding scFv by FACS. The specific selection of the bacteria was demonstrated by amplifying their genes by PCR. CONCLUSIONS: Conditions are described for separating E. coli containing scFv bound to their cell wall by FACS using fluorescently labeled antigens. These studies provide a basis for screening libraries of scFv antibodies.

Amino Acid Sequence↗
PubMed2003

Nucleotide replacement at two sites can be directed by modified single-stranded oligonucleotides in vitro and in vivo.

Studies involving the alteration of DNA sequences by modified single-stranded oligonucleotides in vitro and in vivo have revealed potential applications for functional genomics. Repair of a replacement, deletion, or insertion mutation has already been achieved with molecules having lengths between 25 and 74 bases. But, other vector parameters still remain to be explored. Here, the position of the single base in the vector directing the alteration was examined and the optimal site was found to be at or near the center of the vector. If that position is staggered 3' or 5', the frequencies of gene repair in vitro decreases. The potential of a single vector to direct two nucleotide changes at a specific site in a target sequence was also examined. Both targeted bases are corrected together at the same frequency if the sites are separated by three bases, but conversion linkage decreases precipitously when the distance is expanded to 15 and 27 nucleotides, respectively. These results suggest that single oligonucleotides can be used to direct nucleotide exchange at two independent sites, a reaction characteristic that may be useful for many genomics applications.

Base Pair Mismatch↗
PubMed1986

New plasmid expression vectors for Bacillus subtilis.

The construction of new cloning vectors for Bacillus subtilis is described. They are derived from the in vitro joining of parts of pE194 and pUB110 DNAs. Their common feature is to present a cloning site immediately after the promoter and ribosome binding site of the erythromycin resistance gene, allowing the insertion and expression of either sticky or blunt ended DNA fragments coding for any heterologous gene. The cloning and expression of Escherichia coli beta-lactamase and EcoRI methylase are given as examples. The enzymes are efficiently synthesized by B. subtilis cells.

Bacillus subtilis↗
PubMed1998

Construction of new vectors for high-level expression in actinomycetes.

A new integrative vector (pCJR24) was constructed for use in the erythromycin producer Saccharopolyspora erythraea and in other actinomycetes. It includes the pathway-specific activator gene actII-ORF4 from the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor. The actI promoter and the associated ribosome binding site are located upstream of an NdeI site (5'-CATATG-3') which encompasses the actI start codon allowing protein(s) to be produced at high levels in response to nutritional signals if these signals are faithfully mediated by the ActII-ORF4 activator. Several polyketide synthase genes were cloned in pCJR24 and overexpressed in S. erythraea after integration of the vector into the chromosome by homologous recombination, indicating the possibility that the S. coelicolor promoter/activator functions appropriately in S. erythraea. pCJR24-mediated recombination was also used to place the entire gene set for the erythromycin-producing polyketide synthase under the control of the actI promoter. The resulting strain produced copious quantities of erythromycins and precursor macrolides when compared with wild-type S. erythraea. The use of this system provides the means for rational strain improvement of antibiotic-producing actinomycetes.

Actinomycetales↗
PubMed1987

Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors.

Fragments of exogenous DNA that range in size up to several hundred kilobase pairs have been cloned into yeast by ligating them to vector sequences that allow their propagation as linear artificial chromosomes. Individual clones of yeast and human DNA that have been analyzed by pulsed-field gel electrophoresis appear to represent faithful replicas of the source DNA. The efficiency with which clones can be generated is high enough to allow the construction of comprehensive libraries from the genomes of higher organisms. By offering a tenfold increase in the size of the DNA molecules that can be cloned into a microbial host, this system addresses a major gap in existing experimental methods for analyzing complex DNA sources.

Chromosomes↗
PubMed1997

[Expression of hepatitis C viral structure protein by recombinant vaccinia virus].

The hepatitis C viral (HCV) DNA fragment coding for the core and two glycoproteins E1 + E2/NS1 were inserted into vaccinia virus vector pJSA1175, under the control of the promoter 7.5K. 143TK- cells transfected with the HCV recombinant plasmid pJSA1175CE in the presence of infectious TK+ vaccinia virus (Tiantan stain) yielded recombinant vaccinia virus and expressed an approximately 90 kDa polyprotein. Southern blot hybridization showed that the inserted HCV cDNA was located at the vaccinia virus TK+ gene, J fragment. One polypeptide band between 106 and 80 kDa was revealed in SDS-PAGE which reacted with anti-core murine serum. The polypeptide could be secreted into the medium, the total amount was about the same as in the cells.

Gene Expression↗
PubMedDate not supplied

Introduction of new genetic material into pluripotent haematopoietic stem cells of the mouse.

An infectious retrovirus vector has been used to transfer a bacterial gene encoding resistance to the neomycin analogue G418 into pluripotent haematopoietic stem cells present in explanted murine bone marrow tissue. Subsequent transplantation of the cells into lethally irradiated mice results in engraftment of the animals with donor haematopoietic tissue containing the bacterial gene. This approach affords an efficient and rapid means of re-introducing genetically modified tissue into intact organisms and provides a system whereby the expression and regulation of cloned genes can be followed within the context of a well characterized developmental programme.

Animals↗
PubMed1998

In vivo and in vitro modulation of immune stimulatory capacity of primary dendritic cells by adenovirus-mediated gene transduction.

Dendritic cells (DCs) are potent antigen-presenting cells which are key leukocytes in the initiation of cell-mediated organ graft rejection, antiviral immunity, and antitumor responses. In this study we demonstrate that genetic modification of primary human and mouse DCs by adenoviral gene transfer is an effective means of induction and modulation of antigen presentation by DCs. An adenovirus vector (AdLacZ) was used to express an intracellular model antigen beta-galactosidase (beta-gal) in DCs. Our results show that 30-40% of precursor dendritic cells (PDCs) derived from human umbilical cord blood and circulating mature blood DCs express high levels beta-galactosidase (beta-gal) after infection with AdLacZ with no cytopathic effect observed. In vitro, AdLacZ transduced PDCs and DCs demonstrated a 10- to 20-fold higher mixed lymphocyte reaction (MLR) stimulatory capacity as compared to that of monocytes. In vivo, immunization with AdLacZ transduced mouse DCs resulted in more potent cytotoxic T lymphocyte (CTL) responses against the predicted H-2 restricted beta-gal epitope as compared to CTL responses obtained by beta-gal peptide pulsed DCs. Modulations of the MLR stimulatory capacity of DCs were examined by expression of mouse B7 and CTLA-4Ig. The results show that expression of mouse B7 by a recombinant adenoviral vector (Ad7) significantly enhances the MLR stimulatory capacity of human DCs. In contrast, expression of CTLA-4Ig (AdCTLA-4Ig) reduces the MLR stimulatory capacity of the transduced cells. We conclude that recombinant adenovirus can readily be used for genetic modulation DC-induced immune responses in vivo and in vitro. DCs targeted for induction of specific antigen responses or for modulation of the immune stimulatory capacity may have a potential use in the control of transplantation rejection or viral infections.

Adenoviridae↗
PubMed1994

Phage surface presentation and secretion of antibody fragments using an adaptable phagemid vector.

Phagemid vectors have been developed which promise to supersede hybridoma technology for the selection and production of human antibodies. We have modified an existing phagemid vector to improve the stability of synthesized soluble antibody fragments. The vector allows the antibody fragment to be produced: i) as a soluble protein incorporating a stable carboxyl terminal octapeptide (FLAG) or, ii) on the surface of a bacteriophage fused to a minor coat protein (the gene III protein). The antibody gene encoding the well characterized monoclonal antibody NC10 (an antibody that recognizes the neuraminidase of the influenza strain N9) was inserted as a single chain Fv construct into the phagemid vectors pHFA and pHFA/SacII. Western blotting, ELISA and electron microscopy studies showed that recombinant clones could be manipulated to either synthesize soluble protein into the periplasm or present the protein on the surface of bacteriophage. Cosynthesis of GroEL and GroES chaperonins resulted in complete proteolysis of the scFvNC10-FLAG-gIIIp fusion product and did not improve total phage production. Coexpression of chaperonins should be used with caution for library construction due to the expected selection pressure for protease resistant gene III fusions.

Amino Acid Sequence↗