The Experts below are selected from a list of 82254 Experts worldwide ranked by ideXlab platform
Stephen J Elledge - One of the best experts on this subject based on the ideXlab platform.
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slic a method for sequence and ligation independent cloning
Methods of Molecular Biology, 2012Co-Authors: Stephen J ElledgeAbstract:We describe here a method for sequence- and ligation-independent cloning (SLIC). SLIC uses an exonuclease, T4 DNA polymerase, to generate single-stranded DNA overhangs in insert and vector sequences. These fragments are then assembled in vitro and transformed into Escherichia coli to generate Recombinant DNA of interest. SLIC inserts can also be generated by incomplete PCR (iPCR) or mixed PCR. As many as five inserts can be assembled in one reaction simultaneously with great efficiency using SLIC. SLIC circumvents sequence constraints for Recombinant DNA using standard restriction enzyme-mediated cloning and previous ligation-independent cloning methods and provides a new approach for the efficient generation of Recombinant DNA.
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harnessing homologous recombination in vitro to generate Recombinant DNA via slic
Nature Methods, 2007Co-Authors: Mamie Z Li, Stephen J ElledgeAbstract:We describe a new cloning method, sequence and ligation–independent cloning (SLIC), which allows the assembly of multiple DNA fragments in a single reaction using in vitro homologous recombination and single-strand annealing. SLIC mimics in vivo homologous recombination by relying on exonuclease-generated ssDNA overhangs in insert and vector fragments, and the assembly of these fragments by recombination in vitro. SLIC inserts can also be prepared by incomplete PCR (iPCR) or mixed PCR. SLIC allows efficient and reproducible assembly of Recombinant DNA with as many as 5 and 10 fragments simultaneously. SLIC circumvents the sequence requirements of traditional methods and functions much more efficiently at very low DNA concentrations when combined with RecA to catalyze homologous recombination. This flexibility allows much greater versatility in the generation of Recombinant DNA for the purposes of synthetic biology.
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magic an in vivo genetic method for the rapid construction of Recombinant DNA molecules
Nature Genetics, 2005Co-Authors: Stephen J ElledgeAbstract:We describe a highly engineered in vivo cloning method, mating-assisted genetically integrated cloning (MAGIC), that facilitates the rapid construction of Recombinant DNA molecules. MAGIC uses bacterial mating, in vivo site-specific endonuclease cleavage and homologous recombination to catalyze the transfer of a DNA fragment between a donor vector in one bacterial strain and a recipient plasmid in a separate bacterial strain. Recombination events are genetically selected and result in placement of the gene of interest under the control of new regulatory elements with high efficiency. MAGIC eliminates the need for restriction enzymes, DNA ligases, preparation of DNA and all in vitro manipulations required for subcloning and allows the rapid construction of multiple constructs with minimal effort. We show that MAGIC can generate constructs for expression in multiple organisms. As this new method requires only the simple mixing of bacterial strains, it represents a substantial advance in high-throughput Recombinant DNA production that will save time, effort and expense in functional genomics studies.
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32 rapid construction of Recombinant DNA by the univector plasmid fusion system
Methods in Enzymology, 2000Co-Authors: Qinghua Liu, Dou Liu, Stephen J ElledgeAbstract:Publisher Summary The functional analysis of a single gene often requires many cloning events of the same gene into various vectors for different purposes. Each of these manipulations consumes significant amounts of time and energy for many reasons. First, each gene must be individually tailored for each vector. This is not only because the sequence of every gene is different but also because the majority of existing vectors have been developed independently by different scientists and thus contain different sequences and restriction sites for the insertions of genes. Second, the conventional cut and paste cloning strategy is time-consuming and requires many in vitro manipulations including restriction endonuclease digestion, agrose gel electrophoresis, deoxyribonucleic acid (DNA) fragment isolation, and ligation. Finally, a rational cloning strategy must be designed prior to each cloning event by identifying a compatible vector and suitable restriction enzymes. This normally requires a detailed knowledge of the gene sequence and recipient vector. The advent of the polymerase chain reaction (PCR) and site-directed mutagenesis has greatly facilitated the alteration of gene sequences and the creation of compatible restriction sites for cloning purpose. The high error rate of thermostable polymerases, however, requires each PCR-derived DNA fragment to be verified by DNA sequencing, which is another time-consuming process. To facilitate the rapid, efficient, and uniform construction of Recombinant DNA molecules, a novel cloning strategy, the univector plasmid-fusion system (UPS), has been developed based on the Cre– loxP site-specific recombination system of bacteriophage P1.
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the univector plasmid fusion system a method for rapid construction of Recombinant DNA without restriction enzymes
Current Biology, 1998Co-Authors: Mamie Z Li, Deborah Leibham, David Cortez, Stephen J ElledgeAbstract:Abstract Background: Modern biological research is highly dependent upon Recombinant DNA technology. Conventional cloning methods are time-consuming and lack uniformity. Thus, biological research is in great need of new techniques to rapidly, systematically and uniformly manipulate the large sets of genes currently available from genome projects. Results: We describe a series of new cloning methods that facilitate the rapid and systematic construction of Recombinant DNA molecules. The central cloning method is named the univector plasmid-fusion system (UPS). The UPS uses Cre– lox site-specific recombination to catalyze plasmid fusion between the univector – a plasmid containing the gene of interest – and host vectors containing regulatory information. Fusion events are genetically selected and place the gene under the control of new regulatory elements. A second UPS-related method allows for the precise transfer of coding sequences only from the univector into a host vector. The UPS eliminates the need for restriction enzymes, DNA ligases and many in vitro manipulations required for subcloning, and allows for the rapid construction of multiple constructs for expression in multiple organisms. We demonstrate that UPS can also be used to transfer whole libraries into new vectors. Additional adaptations are described, including directional PCR cloning and the generation of 3′ end gene fusions using homologous recombination in Escherichia coli . Conclusions: Together, these recombination-based cloning methods constitute a new comprehensive approach for the rapid and efficient generation of Recombinant DNA that can be used for parallel processing of large gene sets, a feature that will facilitate future genomic analysis.
Roger A Sedjo - One of the best experts on this subject based on the ideXlab platform.
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far reaching deleterious impacts of regulations on research and environmental studies of Recombinant DNA modified perennial biofuel crops in the united states
BioScience, 2010Co-Authors: Steven H Strauss, Drew L Kershen, Joe H Bouton, Thomas P Redick, Huimin Tan, Roger A SedjoAbstract:Regulatory restrictions have increased in recent years on organisms produced using Recombinant DNA and asexual gene transfer, a process commonly called genetic engineering or genetic modification. Regulatory agencies have raised special concerns and required additional scrutiny for perennial grasses and woody plants of interest for biofuels; these plants have incomplete domestication, invasive capabilities, and the ability to mate with wild or feral relatives. Regulations on these plants require extremely stringent containment through all phases of research and development, regardless of the source of the gene, the novelty of the trait, or the plants' anticipated economic or environmental benefits. We discuss the extent to which these requirements conflict with the realities of practical crop breeding, and prevent meaningful agronomic and environmental studies, thus hampering—and in most cases, precluding—the use of Recombinant DNA breeding methods for perennial crop improvement. We propose regulatory ref...
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far reaching deleterious impacts of regulations on research and environmental studies of Recombinant DNA modified perennial biofuel crops in the united states
Social Science Research Network, 2010Co-Authors: Steven H Strauss, Drew L Kershen, Joe H Bouton, Thomas P Redick, Huimin Tan, Roger A SedjoAbstract:Regulatory restrictions have increased in recent years on organisms produced using Recombinant DNA and asexual gene transfer, a process commonly called genetic engineering or genetic modification. Regulatory agencies have raised special concerns and required additional scrutiny for perennial grasses and woody plants of interest for biofuels; these plants have incomplete domestication, invasive capabilities, and the ability to mate with wild or feral relatives. Regulations on these plants require extremely stringent containment through all phases of research and development, regardless of the source of the gene, the novelty of the trait, or the plants' anticipated economic or environmental benefits. We discuss the extent to which these requirements conflict with the realities of practical crop breeding, and prevent meaningful agronomic and environmental studies, thus hampering — and in most cases, precluding — the use of Recombinant DNA breeding methods for perennial crop improvement. We propose regulatory reforms to better balance benefit and risk and remove unnecessary barriers to agronomic evaluations and environmental studies.
Steven H Strauss - One of the best experts on this subject based on the ideXlab platform.
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far reaching deleterious impacts of regulations on research and environmental studies of Recombinant DNA modified perennial biofuel crops in the united states
BioScience, 2010Co-Authors: Steven H Strauss, Drew L Kershen, Joe H Bouton, Thomas P Redick, Huimin Tan, Roger A SedjoAbstract:Regulatory restrictions have increased in recent years on organisms produced using Recombinant DNA and asexual gene transfer, a process commonly called genetic engineering or genetic modification. Regulatory agencies have raised special concerns and required additional scrutiny for perennial grasses and woody plants of interest for biofuels; these plants have incomplete domestication, invasive capabilities, and the ability to mate with wild or feral relatives. Regulations on these plants require extremely stringent containment through all phases of research and development, regardless of the source of the gene, the novelty of the trait, or the plants' anticipated economic or environmental benefits. We discuss the extent to which these requirements conflict with the realities of practical crop breeding, and prevent meaningful agronomic and environmental studies, thus hampering—and in most cases, precluding—the use of Recombinant DNA breeding methods for perennial crop improvement. We propose regulatory ref...
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far reaching deleterious impacts of regulations on research and environmental studies of Recombinant DNA modified perennial biofuel crops in the united states
Social Science Research Network, 2010Co-Authors: Steven H Strauss, Drew L Kershen, Joe H Bouton, Thomas P Redick, Huimin Tan, Roger A SedjoAbstract:Regulatory restrictions have increased in recent years on organisms produced using Recombinant DNA and asexual gene transfer, a process commonly called genetic engineering or genetic modification. Regulatory agencies have raised special concerns and required additional scrutiny for perennial grasses and woody plants of interest for biofuels; these plants have incomplete domestication, invasive capabilities, and the ability to mate with wild or feral relatives. Regulations on these plants require extremely stringent containment through all phases of research and development, regardless of the source of the gene, the novelty of the trait, or the plants' anticipated economic or environmental benefits. We discuss the extent to which these requirements conflict with the realities of practical crop breeding, and prevent meaningful agronomic and environmental studies, thus hampering — and in most cases, precluding — the use of Recombinant DNA breeding methods for perennial crop improvement. We propose regulatory reforms to better balance benefit and risk and remove unnecessary barriers to agronomic evaluations and environmental studies.
Jacqueline Corrigancuray - One of the best experts on this subject based on the ideXlab platform.
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nih oversight of human gene transfer research involving retroviral lentiviral and adeno associated virus vectors and the role of the nih Recombinant DNA advisory committee
Methods in Enzymology, 2012Co-Authors: Marina Oreilly, Allan C Shipp, Eugene Rosenthal, Robert Jambou, Tom Shih, Maureen Montgomery, Linda Gargiulo, Amy P Patterson, Jacqueline CorrigancurayAbstract:In response to public and scientific concerns regarding human gene transfer research, the National Institutes of Health (NIH) developed a transparent oversight system that extends to human gene transfer protocols that are either conducted with NIH funding or conducted at institutions that receive NIH funding for Recombinant DNA research. The NIH Recombinant DNA Advisory Committee (RAC) has been the primary advisory body to NIH regarding the conduct of this research. Human gene transfer research proposals that are subject to the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines) must be submitted to the NIH Office of Biotechnology Activities (OBA), and protocols that raise novel scientific, safety, medical, ethical, or social issues are publicly discussed at the RAC's quarterly public meetings. OBA also convenes gene transfer safety symposia and policy conferences to provide a public forum for scientific experts to discuss emerging issues in the field. This transparent system of review promotes the rapid exchange of important scientific information and dissemination of data. The goal is to optimize the conduct of individual research protocols and to advance gene transfer research generally. This process has fostered the development of retroviral, lentiviral, and adeno-associated viral vector mediated gene delivery.
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considerations for the clinical application of chimeric antigen receptor t cells observations from a Recombinant DNA advisory committee symposium held june 15 2010
Cancer Research, 2011Co-Authors: Hildegund C J Ertl, John A Zaia, Steven A Rosenberg, Carl H June, Gianpietro Dotti, Jeffrey P Kahn, Laurence J N Cooper, Jacqueline Corrigancuray, Scott E StromeAbstract:T cells that are genetically modified to express single-chain chimeric antigen receptors (CAR) have shown promise in early cancer immunotherapy clinical trials. Unfortunately, 2 recent deaths in cancer patients treated with CAR T cells have created some uncertainty on how to best mitigate patient risk, while continuing to advance this very promising therapeutic avenue. In order to address these concerns, the Recombinant DNA Advisory Committee (RAC) held a symposium, the objectives of which were to first review the reported treatment-associated toxicities and, second, to discuss methods for improving safety and efficacy. This report highlights the issues raised as part of this discussion, with a specific focus on protocols infusing CAR T cells. Because this was not a consensus conference, the opinions described should not be construed to represent those of any individual RAC member, the RAC as a body, conference participants, the National Institutes of Health, or the U.S. Food and Drug Administration.
Qipeng Yuan - One of the best experts on this subject based on the ideXlab platform.
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The Construction of pDH25-pcpC-Vgb as a Recombinant DNA System for the Intracellular Expression of Vitreoscilla Hemoglobin
2016Co-Authors: In Cephalosporium Acremonium, Yubin Liu, Qipeng YuanAbstract:A Recombinant DNA system for the intracellular expression of a bacterial heme-binding protein (Vitreoscilla hemoglobin, Vgb) was constructed and named as pDH25-pcpC-Vgb. It could be introduced into a cephalosporin C-producing strain of Cephalosporium acremonium. The Vgb-expressing transformants will provide higher internal oxygen concentrations, which will cause higher yields of cephalosporin C
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the construction of pdh25 pcpc vgb as a Recombinant DNA system for the intracellular expression of vitreoscilla hemoglobin in cephalosporium acremonium
Mathematical Models and Methods in Applied Sciences, 2009Co-Authors: Yubin Liu, Qipeng YuanAbstract:A Recombinant DNA system for the intracellular expression of a bacterial heme-binding protein (Vitreoscilla hemoglobin, Vgb) was constructed and named as pDH25-pcpC-Vgb. It could be introduced into a cephalosporin C-producing strain of Cephalosporium acremonium. The Vgb-expressing transformants will provide higher internal oxygen concentrations, which will cause higher yields of cephalosporin C.