Agrobacterium Electrocompetent Cells Protocol
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Plant Viral, Agrobacterium Tumefaciens, and Xanthomonas Spp.
USDA May conta in Co nfidential Business Information ~ United States Department of Agricu lture . ... - ------------ --------- - - - ------------ Animal and August 28, 201 4 Plant Health Dr. Luc Mathus Inspection Service Cellectis Plant Sciences 600 County Road D West, Suite 8 Biotechnology Regulatory New Brighton, MN 55112 Servi ces 4700 I iver Road Re: Request for Confirmation that I. ] Potato is not a regulated article Unit 98 Riverdale, MD 20737 Dear Dr. Mathis: Thank you for your letter dated July 29, 2013 inquiring whether or not the potato product described in your letter is a regulated article. This letter states that the "potato has· improved consumer safety and processing attributes attributable to a single gene knock-out achieved through transient expression of a Transcription Activator-Like Effector Nuclease (TALEN)." APHIS regulates the introduction of certain genetically engineered organisms which are, or have the potential to be plant pest. Regulations for genetically engineered organisms that have the potenti al to be plant pests, under the Plant Protection Act, are codified at 7 CFR part 340, "Introduction of Organisms and Products Altered or Produced Through Genetic Engineering Which Are Plant Pest or Which There Is Reason To Believe Are Plant Pests." Under the provisions of these regulations, a genetically engineered (GE) organism is deemed a regulated article if it has been genetically engineered from a donor organism, recipient organism, or vector or vector agent listed in §340.2 and the listed organism meets the definition of "plant pest" or is an unclassified organism and/or an organism whose classification is unknown, or if the Administrator dete11nines that tl e GE organism is a plant pest or has reason to believe is a plant pest. -
Histochemical Analysis of Expression Pattern of Camv 35S Promoter in Transgenic Rapeseed (Brassica Napus L.)
Current World Environment Vol. 10(Special Issue 1), 752-757 (2015) Histochemical Analysis of Expression Pattern of CaMV 35S Promoter in Transgenic Rapeseed (Brassica napus L.) PARISSA JONOUBI1, ALI HateF SALMANIAN2 and AMIN RavaeI3 1Department of Plant Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran. 2Department of Plant Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran. 3Master of Science, Department of Plant Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran. http://dx.doi.org/10.12944/CWE.10.Special-Issue1.90 (Received: November, 2014; Accepted: April, 2015) ABstract Promoters are considered valuable tools for biotechnology and provide great opportunities for eugenic purposes. This study aimed to investigate the expression pattern of GUS gene directed by CaMV 35S promoter in transgenic rapeseed (Brassica napus L.). The GUS gene was transferred to the rapeseed explants by Agrobacterium. The regenerated and rooted transgenic plantlets were transferred to the pots containing a mixture of soli and vermiculite. These plants underwent PCR and histochemical GUS assay. The cross-section of leaf, petiole, and stem of the transformed plants was obtained. GUS activity was observed in phloem, parenchyma, collenchyma, and supporting tissue of vascular bundles. It was also observed in cambium, endoderm, vascular ray, pith parenchyma, epidermis, and trichomes. These results showed that CaMV 35S causes the expression of transgene in various tissues differently. -
World Resources Institute the Monsanto Company
World Resources Institute Sustainable Enterprise Program A program of the World Resources Institute The Monsanto Company: Quest for Sustainability (A) “Biotechnology represents a potentially sustainable For more than a decade, WRI's solution to the issue, not only of feeding people, but of providing Sustainable Enterprise Program (SEP) the economic growth that people are going to need to escape has harnessed the power of business to poverty…… [Biotechnology] poses the possibility of create profitable solutions to leapfrogging the industrial revolution and moving to a post- environment and development industrial society that is not only economically attractive, but challenges. BELL, a project of SEP, is also environmentally sustainable.i ” focused on working with managers and academics to make companies --Robert Shapiro, CEO, Monsanto Company more competitive by approaching social and environmental challenges as unmet market needs that provide Upon his promotion to CEO of chemical giant The business growth opportunities through Monsanto Company in 1995, Robert Shapiro became a vocal entrepreneurship, innovation, and champion of sustainable development and sought to redefine the organizational change. firm’s business strategy along principles of sustainability. Shapiro’s rhetoric was compelling. He captured analysts’ Permission to reprint this case is attention with the specter of mass hunger and environmental available at the BELL case store. degradation precipitated by rapid population growth and the Additional information on the Case -
The Ethics of Changing People Through Genetic Engineering, 13 Notre Dame J.L
Notre Dame Journal of Law, Ethics & Public Policy Volume 13 Article 5 Issue 1 Sym[posium on Emerging Issues in Technology 1-1-2012 What Sort of People Do We Want - The thicE s of Changing People through Genetic Engineering Michael J. Reiss Follow this and additional works at: http://scholarship.law.nd.edu/ndjlepp Recommended Citation Michael J. Reiss, What Sort of People Do We Want - The Ethics of Changing People through Genetic Engineering, 13 Notre Dame J.L. Ethics & Pub. Pol'y 63 (1999). Available at: http://scholarship.law.nd.edu/ndjlepp/vol13/iss1/5 This Article is brought to you for free and open access by the Notre Dame Journal of Law, Ethics & Public Policy at NDLScholarship. It has been accepted for inclusion in Notre Dame Journal of Law, Ethics & Public Policy by an authorized administrator of NDLScholarship. For more information, please contact [email protected]. WHAT SORT OF PEOPLE DO WE WANT? THE ETHICS OF CHANGING PEOPLE THROUGH GENETIC ENGINEERING MICHAEL J. REISS* Within the last decade, genetic engineering has changed from being a relatively esoteric research technique of molecular biologists to an application of considerable power, yet one which raises widespread public concern. In this article, I first briefly summarize the principles of genetic engineering, as applied to any organism. I then concentrate on humans, reviewing both progress to date and possible future developments. Throughout, my particular focus is on the ethical acceptability or otherwise of the technology. I restrict myself to cases where humans are themselves being genetically engineered. This means, for instance, that I do not cover such topics as xenotransplantation (when animals are genetically engineered to make them suitable for transplantation into humans) and the issues resulting from the production of such products as genetically engineered human growth hor- mone, al-antitrypsin, or vaccines (when micro-organisms, ani- mals, or plants are genetically engineered to produce human proteins). -
Quick and Efficient Method for Genetic Transformation of Biopolymer
Technical Note Received: 29 July 2009 Revised: 14 September 2009 Accepted: 14 September 2009 Published online in Wiley Interscience: 29 October 2009 (www.interscience.wiley.com) DOI 10.1002/jctb.2284 Quick and efficient method for genetic transformation of biopolymer-producing bacteria Qin Wang,a Alexander P. Mueller,a Chean Ring Leong,b Ken’ichiro Matsumoto,b Seiichi Taguchib and Christopher T. Nomuraa∗ Abstract In order to genetically modify microorganisms capable of producing polyhydroxyalkanoate (PHA) biopolymers, a simple and rapid method to prepare freshly plated Pseudomonas cells for transformation via electroporation was developed. This method can be used to transfer both replicative plasmids and linear DNA to knock out genes into the cells. The transformation efficiencies were in the range of ≥107 transformants µg−1 DNA for replicative plasmids and ≥106 transformants µg−1 DNA for linear DNA, which are comparable with commercially available competent cells. Furthermore, this transformation procedure can be performed in less than 10 min, saving a great deal of time compared with traditional methods. Knockout mutants of several Pseudomonas species were generated by transformation of linear DNA and these mutations were verified by PCR and analysis of PHA content. c 2009 Society of Chemical Industry Keywords: transformation; electroporation; Pseudomonas putida; polyhydroxyalkanoates (PHAs) INTRODUCTION using various strains of P. putida.StrainsweregrowninLuria- Pseudomonas putida is a Gram-negative soil bacterium that plays Bertani (LB) medium (1% tryptone, 0.5% yeast extract, and 0.5% animportantroleinelementcycling innature,bioremediation,and NaCl) with the appropriate antibiotic when necessary. For selection production of polyhydroxyalkanoates (PHAs), which are environ- of transformants, kanamycin (Km) and gentamycin (Gm) were mentally friendly biodegradable plastics.1–3 Despite having a fully added to LB agar plates and liquid media at final concentrations sequenced genome,3 the functions of many ORFs in this organ- of 50 µgmL−1 and 20 µgmL−1, respectively. -
ES Cell Targeting Handbook
TABLE OF CONTENTS Overview of ES Core Facility Introduction Generation of Gene-Targeted ES Cells Karyotyping of Positive ES Clones ES Cell Request Form General Information for the Generation of Targeted Cells Principles of Gene Targeting Requirements for the Design of Targeting Constructs Screening Assay for the Identification of Targeted ES Clones Overview of ES Cell Culture ES Cell Factors Affecting Successful Chimera Production FAQ Overview of ES Core Facility Our Mission The ES Core Facility (ECF) was founded by the NINDS Core Center Grant and was established to benefit the contributors of this proposal. The mission of ECF is to effectively produce ES cell lines with a high probability of germline transmission. Core Service Services provided by the Core for a typical project include: • Provide guidance on the design of targeting construct • Generate targeted ES cell lines for the production of chimeric mice • Karyotyping ES cells to be micro-injected into blastocysts Consultation is available from ECF directors and staff members on the entire procedures of generating gene knock-out mice. Application for Service Prior to the initiation of a project, a brief meeting is generally required between the investigator and ECF facility staff resulting in a mutually acceptable research strategy. This strategy will outline specifics of the project including knockout strategy, KO construct design, screening assays, and other procedural issues relevant to the generation of targeted ES cells. In addition, a completed service application form, signed by the principal investigator and approved by the Core Director, will also be required. The Core Director will prioritize the service requests according to the difficulty of the project and work load. -
Intramuscular Electroporation Delivery of IFN- Gene Therapy for Inhibition of Tumor Growth Located at a Distant Site
Gene Therapy (2001) 8, 400–407 2001 Nature Publishing Group All rights reserved 0969-7128/01 $15.00 www.nature.com/gt RESEARCH ARTICLE Intramuscular electroporation delivery of IFN-␣ gene therapy for inhibition of tumor growth located at a distant site S Li, X Zhang, X Xia, L Zhou, R Breau, J Suen and E Hanna Department of Otolaryngology/Head and Neck Surgery, University of Arkansas School of Medicine, 4001 W Capital Avenue, Little Rock, AR 72205, USA Although electroporation has been shown in recent years to 2 or endostatin gene, also delivered by electro-injection. The be a powerful method for delivering genes to muscle, no increased therapeutic efficacy was associated with a high gene therapy via electro-injection has been studied for the level and extended duration of IFN-␣ expression in muscle treatment of tumors. In an immunocompetent tumor-bearing and serum. We also discovered that the high level of IFN-␣ murine model, we have found that delivery of a low dose of expression correlated with increased expression levels of reporter gene DNA (10 g) to muscle via electroporation the antiangiogenic genes IP-10 and Mig in local tumor under specific pulse conditions (two 25-ms pulses of 375 tissue, which may have led to the reduction of blood vessels V/cm) increased the level of gene expression by two logs of observed at the local tumor site. Delivery of increasing doses magnitude. Moreover, administration of 10 g of interferon (10–100 g) of IFN-␣ plasmid DNA by injection alone did (IFN)-␣ DNA plasmid using these parameters once a week not increase antitumor activity, whereas electroporation for 3 weeks increased the survival time and reduced squam- delivery of increasing doses (10–40 g) of IFN-␣ plasmid ous cell carcinoma (SCC) growth at a distant site in the DNA did increase the survival time. -
Import of DNA Into Mammalian Nuclei by Proteins Originating from a Plant Pathogenic Bacterium
Proc. Natl. Acad. Sci. USA Vol. 96, pp. 3729–3733, March 1999 Cell Biology Import of DNA into mammalian nuclei by proteins originating from a plant pathogenic bacterium i ALICJA ZIEMIENOWICZ*, DIRK GO¨RLICH†,ERICH LANKA§,BARBARA HOHN*¶, AND LUCA ROSSI* *Friedrich Miescher-Institut, P.O. Box 2543, CH-4002 Basel, Switzerland; †Zentrum fu¨r Molekulare Biologie der Universita¨tHeidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany; and §Max-Planck Institut fu¨r Molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Germany Communicated by Diter von Wettstein, Washington State University, Pullman, WA, February 2, 1999 (received for review October 22, 1998) ABSTRACT Import of DNA into mammalian nuclei is accompanying it into the plant. The bacterial proteins VirD2 generally inefficient. Therefore, one of the current challenges and VirE2 both contain bipartite nuclear localization signals in human gene therapy is the development of efficient DNA (NLSs) that target them into plant nuclei (7–9). Furthermore, delivery systems. Here we tested whether bacterial proteins the C-terminal NLS of the VirD2 protein is required for could be used to target DNA to mammalian cells. Agrobacte- efficient transfer of the bacterial T-DNA to the plant nucleus rium tumefaciens, a plant pathogen, efficiently transfers DNA (10–12). Because mammalian NLSs have been shown to be as a nucleoprotein complex to plant cells. Agrobacterium- recognized in plant systems (13–16), these target sequences mediated T-DNA transfer to plant cells is the only known may be universal. example for interkingdom DNA transfer and is widely used for We reconstituted in vitro the T-DNA complexes composed plant transformation. -
This Document Is the Property of Bayer AG And/Or
Safety Assessment Summary of Genuity Roundup Ready 2Yield MON 89788 Soybean Executive Summary Ongoing developments in biotechnology and molecular-assisted breeding have enabled Monsanto to develop a second-generation glyphosate-tolerant soybean product: GenuityTM Roundup Ready 2 Yield® or MON 89788 soybean (OECD Unique ID: MON–89788–1). Similar to the first generation product Roundup Ready® soybean, MON 89788 soybean will continue to provide growers with flexibility, simplicity, and cost effective weed control options. However, MON 89788 soybean and varieties containing the trait have the added potential toand and regime. enhance yield and thereby further benefit farmersAG and the soybean industry. In 1996, Roundup Ready soybean was the first soybean product containing a biotechnology trait to be commercialized in the U.S. Roundup Ready soybean was produced by incorporation of the cp4 epspsBayer coding property sequence derivedpublishing from the protectioncontents common soil bacterium Agrobacteriumof sp. strain CP4. The cp4 epsps coding its sequence directs the production of the 5-enolpyruvylparties. datashikimate -3-phosphatetherefore and/oror synthase (termed CP4 EPSPS) that is much less sensitive to glyphosate inhibition affiliates. than endogenous plant EPSPS.property The CP4 EPSPSintellectualthird renders Roundup Readymay soybean its tolerant to glyphosate, which is the asactive ingredient in Roundup agricultural the of and herbicides. The utilizationis of Roundup agriculturalregulatory herbicides plusowner. Round up a document -
Barley Transformation Using Agrobacterium-Mediated Techniques
Barley Transformation using Agrobacterium-Mediated Techniques Wendy A Harwood, Joanne G Bartlett, Silvia C Alves, Matthew Perry, Mark A Smedley, Nicola Leyland and John W Snape Abstract Methods for the transformation of barley using Agrobacterium-mediated techniques have been available for the past ten years. Agrobacterium offers a number of advantages over Biolistic-mediated techniques in terms of efficiency and the quality of the transformed plants produced. This chapter describes a simple system for the transformation of barley based on the infection of immature embryos with Agrobacterium tumefaciens followed by the selection of transgenic tissue on media containing the antibiotic hygromycin. The method can lead to the production of large numbers of fertile, independent transgenic lines. It is therefore ideal for studies of gene function in a cereal crop system. Key Words: Barley transformation, Agrobacterium tumefaciens, transgenic plants, hygromycin, immature embryo. 1 1. Introduction The first reports of successful barley transformation (1) used biolistic-based techniques to introduce DNA to immature embryos. Immature embryos were also the target tissue used in the first reports of the generation of transgenic barley plants using Agrobacterium (2). Although alternative target tissues have been examined for use in barley transformation systems, immature embryos remain the target tissue of choice for obtaining high transformation efficiencies. An alternative Agrobacterium- mediated barley transformation system uses microspore cultures as the target tissue (3). A comparison of biolistic and Agrobacterium-based methods for barley transformation highlighted some of the advantages of the Agrobacterium system (4). These included higher transformation efficiencies, lower transgene copy number and more stable inheritance of the transgenes with less transgene silencing. -
Micropropagation, Genetic Engineering, and Molecular Biology of Populus
This file was created by scanning the printed publication. Errors identified by the software have been corrected; however, some errors may remain. Chapter7 Agrobacterium-mediated Transformation of Populus Species1 Mee-Sook Kim, Ned B. Klopfenstein, and Young Woo Chun posed by wounding (Perani et al. 1986). Infection by A. Introduction tumefaciens causes crown gall disease (figure 1), whereas A. rhizogenes causes hairy root disease. In addition to its chromosomal DNA, Agrobacterium contains 2 other genetic Although molecular biology of woody plants is a rela components that are required for plant cell transforma tively young field, it offers considerable potential for breed tion; T-DNA (transferred DNA) and the virulence (vir) re ing and selecting improved trees for multiple purposes. gion, which are both located on the TI (tumor-inducing) or Conventional breeding programs have produced im Ri (root-inducing) plasmid (Zambryoski et al. 1989). The proved growth rates, adaptability, and pest resistance; T-DNA portion of the A. tumefaciens TI plasmid or the A. however, tree improvement processes are time consum rhizogenes Ri plasmid is transferred to the nucleus of a host ing because of the long generation and rotation cycles of plant where it integrates into the nuclear DNA genetically trees (Dinus and Tuskan this volume; Leple et al. 1992). transforming the recipient plant. A region of the 1i plas Genetic engineering of trees helps to compensate for con mid outside the T-DNA, referred to as the wirulence re ventional breeding disadvantages by incorporating known gion, carries the vir genes. Expression of vir genes occurs genes into specific genetic backgrounds. -
Engineering of Primary Human B Cells with CRISPR/Cas9 Targeted Nuclease Received: 26 January 2018 Matthew J
www.nature.com/scientificreports OPEN Engineering of Primary Human B cells with CRISPR/Cas9 Targeted Nuclease Received: 26 January 2018 Matthew J. Johnson1,2,3, Kanut Laoharawee1,2,3, Walker S. Lahr1,2,3, Beau R. Webber1,2,3 & Accepted: 23 July 2018 Branden S. Moriarity1,2,3 Published: xx xx xxxx B cells ofer unique opportunities for gene therapy because of their ability to secrete large amounts of protein in the form of antibody and persist for the life of the organism as plasma cells. Here, we report optimized CRISPR/Cas9 based genome engineering of primary human B cells. Our procedure involves enrichment of CD19+ B cells from PBMCs followed by activation, expansion, and electroporation of CRISPR/Cas9 reagents. We are able expand total B cells in culture 10-fold and outgrow the IgD+ IgM+ CD27− naïve subset from 35% to over 80% of the culture. B cells are receptive to nucleic acid delivery via electroporation 3 days after stimulation, peaking at Day 7 post stimulation. We tested chemically modifed sgRNAs and Alt-R gRNAs targeting CD19 with Cas9 mRNA or Cas9 protein. Using this system, we achieved genetic and protein knockout of CD19 at rates over 70%. Finally, we tested sgRNAs targeting the AAVS1 safe harbor site using Cas9 protein in combination with AAV6 to deliver donor template encoding a splice acceptor-EGFP cassette, which yielded site-specifc integration frequencies up to 25%. The development of methods for genetically engineered B cells opens the door to a myriad of applications in basic research, antibody production, and cellular therapeutics.